Genes and polypeptides relating to human colon cancers

ABSTRACT

The present application provides novel human genes RNF43 whose expression is markedly elevated in colorectal cancers, as well as CXADRL1 and GCUD1 whose expression is markedly elevated in gastric cancers compared to corresponding non-cancerous tissues. The genes and polypeptides encoded by the genes can be used, for example, in the diagnosis of a cell proliferative disease, and as target molecules for developing drugs against the disease.

The present application is a divisional of U.S. Ser. No. 10/916,064,filed Aug. 10, 2004, which is a continuation in part ofPCT/JP2003/007006, filed Jun. 3, 2003, which claims priority to U.S.Ser. No. 60/386,985, filed Jun. 6, 2002, U.S. Ser. No. 60/415,209, filedSep. 30, 2002, and U.S. Ser. No. 60/451,013, filed Feb. 28, 2003, eachof which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer research. In particular, the presentinvention relates to novel genes, RNF43, CXADRL1, and GCUD1, involved inthe proliferation mechanism of cells, as well as polypeptides encoded bythe genes. The genes and polypeptides of the present invention can beused, for example, in the diagnosis of cell proliferative disease, andas target molecules for developing drugs against the disease.

BACKGROUND ART

Gastric cancers and colorectal cancers are leading causes of cancerdeath worldwide. In spite of recent progress in diagnostic andtherapeutic strategies, prognosis of patients with advanced cancersremains very poor. Although molecular studies have revealed theinvolvement of alterations in tumor suppressor genes and/or oncogenes incarcinogenesis, the precise mechanisms still remain to be elucidated.

cDNA microarray technologies have enabled to obtain comprehensiveprofiles of gene expression in normal and malignant cells, and comparethe gene expression in malignant and corresponding normal cells (Okabeet al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al.,Cancer Res 62:7012-7 (2002)). This approach enables to disclose thecomplex nature of cancer cells, and helps to understand the mechanism ofcarcinogenesis. Identification of genes that are deregulated in tumorscan lead to more precise and accurate diagnosis of individual cancers,and to develop novel therapeutic targets (Bienz and Clevers, Cell103:311-20 (2000)). To disclose mechanisms underlying tumors from agenome-wide point of view, and discover target molecules for diagnosisand development of novel therapeutic drugs, the present inventors havebeen analyzing the expression profiles of tumor cells using cDNAmicroarray of 23040 genes (Okabe et al., Cancer Res 61:2129-37 (2001);Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)).

Studies designed to reveal mechanisms of carcinogenesis have alreadyfacilitated identification of molecular targets for anti-tumor agents.For example, inhibitors of farnesyltransferase (FTIs) which wereoriginally developed to inhibit the growth-signaling pathway related toRas, whose activation depends on posttranslational farnesylation, hasbeen effective in treating Ras-dependent tumors in animal models (He etal., Cell 99:335-45 (1999)). Clinical trials on human using acombination of anti-cancer drugs and anti-HER2 monoclonal antibody,trastuzumab, have been conducted to antagonize the proto-oncogenereceptor HER2/neu; and have been achieving improved clinical responseand overall survival of breast-cancer patients (Lin et al, Cancer Res61:6345-9 (2001)). A tyrosine kinase inhibitor, STI-571, whichselectively inactivates bcr-abl fusion proteins, has been developed totreat chronic myelogenous leukemias wherein constitutive activation ofbcr-abl tyrosine kinase plays a crucial role in the transformation ofleukocytes. Agents of these kinds are designed to suppress oncogenicactivity of specific gene products (Fujita et al., Cancer Res 61:7722-6(2001)). Therefore, gene products commonly up-regulated in cancerouscells may serve as potential targets for developing novel anti-canceragents.

It has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs)recognize epitope peptides derived from tumor-associated antigens (TAAs)presented on MHC Class I molecule, and lyse tumor cells. Since thediscovery of MAGE family as the first example of TAAs, many other TAAshave been discovered using immunological approaches (Boon, Int J Cancer54: 177-80 (1993); Boon and van der Bruggen, J Exp Med 183: 725-9(1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard etal., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180:347-52 (1994)). Some of the discovered TAAs are now in the stage ofclinical development as targets of immunotherapy. TAAs discovered so farinclude MAGE (van der Bruggen et al., Science 254: 1643-7 (1991)), gp100(Kawakami et al., J Exp Med 180: 347-52 (1994)), SART (Shichijo et al.,J Exp Med 187: 277-88 (1998)), and NY-ESO-1 (Chen et al., Proc Natl AcadSci USA 94: 1914-8 (1997)). On the other hand, gene products which hadbeen demonstrated to be specifically overexpressed in tumor cells, havebeen shown to be recognized as targets inducing cellular immuneresponses. Such gene products include p53 (Umano et al., Brit J Cancer84: 1052-7 (2001)), HER2/neu (Tanaka et al., Brit J Cancer 84: 94-9(2001)), CEA (Nukaya et al., Int J Cancer 80: 92-7 (1999)), and so on.

In spite of significant progress in basic and clinical researchconcerning TAAs (Rosenbeg et al., Nature Med 4: 321-7 (1998); Mukherjiet al., Proc Natl Acad Sci USA 92: 8078-82 (1995); Hu et al., Cancer Res56: 2479-83 (1996)), only limited number of candidate TAAs for thetreatment of adenocarcinomas, including colorectal cancer, areavailable. TAAs abundantly expressed in cancer cells, and at the sametime which expression is restricted to cancer cells would be promisingcandidates as immunotherapeutic targets. Further, identification of newTAAs inducing potent and specific antitumor immune responses is expectedto encourage clinical use of peptide vaccination strategy in varioustypes of cancer (Boon and can der Bruggen, J Exp Med 183: 725-9 (1996);van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., JExp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52(1994); Shichijo et al., J Exp Med 187: 277-88 (1998); Chen et al., ProcNatl Acad Sci USA 94: 1914-8 (1997); Harris, J Natl Cancer Inst 88:1442-5 (1996); Butterfield et al., Cancer Res 59: 3134-42 (1999);Vissers et al., Cancer Res 59: 5554-9 (1999); van der Burg et al., JImmunol 156: 3308-14 (1996); Tanaka et al., Cancer Res 57: 4465-8(1997); Fujie et al., Int J Cancer 80: 169-72 (1999); Kikuchi et al.,Int J Cancer 81: 459-66 (1999); Oiso et al., Int J Cancer 81: 387-94(1999)).

It has been repeatedly reported that peptide-stimulated peripheral bloodmononuclear cells (PBMCs) from certain healthy donors producesignificant levels of IFN-γ in response to the peptide, but rarely exertcytotoxicity against tumor cells in an HLA-A24 or -A0201 restrictedmanner in ⁵¹Cr-release assays (Kawano et al., Cancer Res 60: 3550-8(2000); Nishizaka et al., Cancer Res 60: 4830-7 (2000); Tamura et al.,Jpn J Cancer Res 92: 762-7 (2001)). However, both of HLA-A24 andHLA-A0201 are one of the popular HLA alleles in Japanese, as well asCaucasian (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al.,J Immunol 155: 4307-12 (1995); Kubo et al., J Immunol 152: 3913-24(1994); Imanishi et al., Proceeding of the eleventh InternationalHictocompatibility Workshop and Conference Oxford University Press,Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)).Thus, antigenic peptides of cancers presented by these HLAs may beespecially useful for the treatment of cancers among Japanese andCaucasian. Further, it is known that the induction of low-affinity CTLin vitro usually results from the use of peptide at a highconcentration, generating a high level of specific peptide/MHC complexeson antigen presenting cells (APCs), which will effectively activatethese CTL (Alexander-Miller et al., Proc Natl Acad Sci USA 93: 4102-7(1996)).

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel proteins involvedin the proliferation mechanism of gastric or colorectal cancer cells andthe genes encoding the proteins, as well as methods for producing andusing the same in the diagnosis and treatment of gastric cancer orcolorectal cancer.

To disclose the mechanism of gastric and colorectal carcinogenesis andidentify novel diagnostic markers and/or drug targets for the treatmentof these tumors, the present inventors analyzed the expression profilesof genes in gastric and colorectal carcinogenesis using a genome-widecDNA microarray containing 23040 genes. From the pharmacological pointof view, suppressing oncogenic signals is easier in practice thanactivating tumor-suppressive effects. Thus, the present inventorssearched for genes that are up-regulated during gastric and colorectalcarcinogenesis.

Among the transcripts that were commonly up-regulated in gastriccancers, novel human genes CXADRL1 (coxsackie and adenovirus receptorlike 1) and GCUD1 (up-regulated in gastric cancer) were identified onchromosome band 3q13 and 7p14, respectively. Gene transfer of CXADRL1 orGCUD1 promoted proliferation of cells. Furthermore, reduction of CXADRL1or GCUD1 expression by transfection of their specific antisenseS-oligonucleotides or small interfering RNAs inhibited the growth ofgastric cancer cells. Many anticancer drugs, such as inhibitors of DNAand/or RNA synthesis, metabolic suppressors, and DNA intercalators, arenot only toxic to cancer cells but also for normally growing cells.However, agents suppressing the expression of CXADRL1 may not adverselyaffect other organs due to the fact that normal expression of the geneis restricted to the testis and ovary, and thus may be of greatimportance for treating cancer.

Furthermore, among the transcripts that were commonly up-regulated incolorectal cancers, gene RNF43 (Ring finger protein 43) assigned atchromosomal band 17pter-p13.1 was identified. In addition, yeasttwo-hybrid screening assay revealed that RNF43 protein associated withNOTCH2 or STRIN.

NOTCH2 is a large transmembrane receptor protein that is a component ofan evolutionarily conserved intercellular signaling mechanism. NOTCH2 isa protein member of the Notch signaling pathway and is reported to beinvolved in glomerulogenesis in the kidney and development of heart andeye vasculature (McCright et al., Development 128: 491-502 (2001)).Three Delta/Serrate/Lag-2 (DSL) proteins, Delta1, Jaggaed1, andJaggaed2, are reported as functional ligands for NOTCH2 (Shimizu et al.,Mol Cell Biol 20: 6913-22 (2000)). The signal induced by ligand bindingin the Notch signaling pathway is transmitted intracellulaly by aprocess involving proteolysis of the receptor and nuclear translocationof the intracellular domain of the NOTCH protein (see reviewsArtavanis-Tsakonas et al., Annu Rev Cell Biol 7: 427-52 (1999);Weinmaster, Curr Opin Genet Dev 10: 363-9 (2000)). Furthermore,reduction of RNF43 expression by transfection of specific antisenseS-oligonucleotides or small interfering RNAs corresponding to RNF43inhibited the growth of colorectal cancer cells. As already describedabove many anticancer drugs, are not only toxic to cancer cells but alsofor normally growing cells. However, agents suppressing the expressionof RNF43 may also not adversely affect other organs due to the fact thatnormal expression of the gene is restricted to fetus, more specificallyfetal lung and fetal kidney, and thus may be of great importance fortreating cancer.

Thus, the present invention provides isolated novel genes, CXADRL1,GCUD1, and RNF43, which are candidates as diagnostic markers for canceras well as promising potential targets for developing new strategies fordiagnosis and effective anti-cancer agents. Furthermore, the presentinvention provides polypeptides encoded by these genes, as well as theproduction and the use of the same. More specifically, the presentinvention provides the following:

The present application provides novel human polypeptides, CXADRL1,GCUD1, and RNF43, and functional equivalents thereof, that promote cellproliferation and is up-regulated in cell proliferative diseases, suchas gastric and colorectal cancers.

In a preferred embodiment, the CXADRL1 polypeptide includes a putative431 amino acid protein with about 37% identity to CXADR (coxsackie andadenovirus receptor). CXADRL1 is encoded by the open reading frame ofSEQ ID NO: 1 and contains two immunogloblin domains at codons 29-124 and158-232, as well as a transmembrane domain at codons 246-268. TheCXADRL1 polypeptide preferably includes the amino acid sequence setforth in SEQ ID NO: 2. The present application also provides an isolatedprotein encoded from at least a portion of the CXADRL1 polynucleotidesequence, or polynucleotide sequences at least 30%, more preferably atleast 40% complementary to the sequence set forth in SEQ ID NO: 1.

On the other hand, in a preferred embodiment, the GCUD1 polypeptideincludes a putative 414 amino acid protein encoded by the open readingframe of SEQ ID NO: 3. The GCUD1 polypeptide preferably includes theamino acid sequence set forth in SEQ ID NO: 4. The present applicationalso provides an isolated protein encoded from at least a portion of theGCUD1 polynucleotide sequence, or polynucleotide sequences at least 15%,more preferably at least 25% complementary to the sequence set forth inSEQ ID NO: 3.

Furthermore, in a preferred embodiment, the RNF43 polypeptide includes aputative 783 amino acid protein encoded by the open reading frame of SEQID NO: 5. The RNF43 polypeptide preferably includes the amino acidsequence set forth in SEQ ID NO: 6 and contains a Ring finger motif atcodons 272-312. The RNF43 polypeptide showed 38% homology to RING fingerprotein homolog DKFZp566H073.1 (GenBank Accession Number: T08729). Thepresent application also provides an isolated protein encoded from atleast a portion of the RNF43 polynucleotide sequence, or polynucleotidesequences at least 30%, more preferably at least 40% complementary tothe sequence set forth in SEQ ID NO: 5.

The present invention further provides novel human genes, CXADRL1 andGCUD1, whose expressions are markedly elevated in a great majority ofgastric cancers as compared to corresponding non-cancerous mucosae. Inaddition to gastric cancers, CXADRL1 and GCUD1 were also highlyexpressed in colorectal cancer and liver cancer. The isolated CXADRL1gene includes a polynucleotide sequence as described in SEQ ID NO: 1. Inparticular, the CXADRL1 cDNA includes 3423 nucleotides that contain anopen reading frame of 1296 nucleotides (SEQ ID NO: 1). The presentinvention further encompasses polynucleotides which hybridize to andwhich are at least 30%, and more preferably at least 40% complementaryto the polynucleotide sequence set forth in SEQ ID NO: 1, to the extentthat they encode a CXADRL1 protein or a functional equivalent thereof.Examples of such polynucleotides are degenerates and allelic mutants ofSEQ ID NO: 1. On the other hand, the isolated GCUD1 gene includes apolynucleotide sequence as described in SEQ ID NO: 3. In particular, theGCUD1 cDNA includes 4987 nucleotides that contain an open reading frameof 1245 nucleotides (SEQ ID NO: 3). The present invention furtherencompasses polynucleotides which hybridize to and which are at least15%, and more preferably at least 25% complementary to thepolynucleotide sequence set forth in SEQ ID NO: 3, to the extent thatthey encode a GCUD1 protein or a functional equivalent thereof. Examplesof such polynucleotides are degenerates and allelic mutants of SEQ IDNO: 3.

Furthermore, the present invention provides a novel human gene RNF43,whose expression is markedly elevated in a great majority of colorectalcancers as compared to corresponding non-cancerous mucosae. In additionto colorectal cancers, RNF43 was also highly expressed in lung cancer,gastric cancer, and liver cancer. The isolated RNF43 gene includes apolynucleotide sequence as described in SEQ ID NO: 5. In particular, theRNF43 cDNA includes 5345 nucleotides that contain an open reading frameof 2352 nucleotides (SEQ ID NO: 5). The present invention furtherencompasses polynucleotides which hybridize to and which are at least30%, and more preferably at least 40% complementary to thepolynucleotide sequence set forth in SEQ ID NO: 5, to the extent thatthey encode a RNF43 protein or a functional equivalent thereof. Examplesof such polynucleotides are degenerates and allelic mutants of SEQ IDNO: 5.

As used herein, an isolated gene is a polynucleotide whose structure isnot identical to that of any naturally occurring polynucleotide or tothat of any fragment of a naturally occurring genomic polynucleotidespanning more than three separate genes. The term therefore includes,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule in the genome of the organism in which itnaturally occurs; (b) a polynucleotide incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion polypeptide.

Accordingly, in one aspect, the invention provides an isolatedpolynucleotide that encodes a polypeptide described herein or a fragmentthereof. Preferably, the isolated polypeptide includes a nucleotidesequence that is at least 60% identical to the nucleotide sequence shownin SEQ ID NO: 1, 3, or 5. More preferably, the isolated nucleic acidmolecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequenceshown in SEQ ID NO: 1, 3, or 5. In the case of an isolatedpolynucleotide which is longer than or equivalent in length to thereference sequence, e.g., SEQ ID NO: 1, 3, or 5, the comparison is madewith the full-length of the reference sequence. When the isolatedpolynucleotide is shorter than the reference sequence, e.g., shorterthan SEQ ID NO: 1, 3, or 5, the comparison is made to segment of thereference sequence of the same length (excluding any loop required bythe homology calculation).

The present invention also provides a method of producing a protein bytransfecting or transforming a host cell with a polynucleotide sequenceencoding the CXADRL1, GCUD1, or RNF43 protein, and expressing thepolynucleotide sequence. In addition, the present invention providesvectors comprising a nucleotide sequence encoding the CXADRL1, GCUD1, orRNF43 protein, and host cells harboring a polynucleotide encoding theCXADRL1, GCUD1, or RNF43 protein. Such vectors and host cells may beused for producing the CXADRL1, GCUD1, or RNF43 protein.

An antibody that recognizes the CXADRL1, GCUD1, or RNF43 protein is alsoprovided by the present application. In part, an antisensepolynucleotide (e.g., antisense DNA), ribozyme, and siRNA (smallinterfering RNA) of the CXADRL1, GCUD1, or RNF43 gene is also provided.

The present invention further provides a method for diagnosis of cellproliferative diseases that includes determining an expression level ofthe gene in biological sample of specimen, comparing the expressionlevel of CXADRL1, GCUD1, or RNF43 gene with that in normal sample, anddefining a high expression level of the CXADRL1, GCUD1, or RNF43 gene inthe sample as having a cell proliferative disease such as cancer. Thedisease diagnosed by the expression level of CXADRL1 or GCUD1 issuitably a gastric, colorectal, and liver cancer; and that detected bythe expression level of RNF43 is colorectal, lung, gastric, and livercancer.

Furthermore, a method of screening for a compound for treating a cellproliferative disease is provided. The method includes the steps ofcontacting the CXADRL1, GCUD1, or RNF43 polypeptide with test compounds,and selecting test compounds that bind to the CXADRL1, GCUD1, or RNF43polypeptide.

The present invention further provides a method of screening for acompound for treating a cell proliferative disease, wherein the methodincludes the steps of contacting the CXADRL1, GCUD1, or RNF43polypeptide with a test compound, and selecting the test compound thatsuppresses the expression level or biological activity of the CXADRL1,GCUD1, or RNF43 polypeptide.

Alternatively, the present invention provides a method of screening fora compound for treating a cell proliferative disease, wherein the methodincludes the steps of contacting CXADRL1 and AIP1 in the presence of atest compound, and selecting the test compound that inhibits the bindingof CXADRL1 and AIP1.

Furthermore, the present invention provides a method of screening for acompound for treating a cell proliferative disease, wherein the methodincludes the steps of contacting RNF43 and NOTCH2 or STRIN in thepresence of a test compound, and selecting the test compound thatinhibits the binding of RNF43 and NOTCH2 or STRIN.

The present application also provides a pharmaceutical composition fortreating cell proliferative disease, such as cancer. The pharmaceuticalcomposition may be, for example, an anti-cancer agent. Thepharmaceutical composition can be described as at least a portion of theantisense S-oligonucleotides or siRNA of the CXADRL1, GCUD1, or RNF43polynucleotide sequence shown and described in SEQ ID NO: 1, 3, or 5,respectively. A suitable antisense S-oligonucleotide has the nucleotidesequence selected from the group of SEQ ID NO: 23, 25, 27, 29, or 31.The antisense S-oligonucleotide of CXADRL1 including those having thenucleotide sequence of SEQ ID NO: 23 or 25 may be suitably used to treatgastric, colorectal and liver cancer; the antisense S-oligonucleotide ofGCUD1 including those having the nucleotide sequence of SEQ ID NO: 27 or29, suitably to treat gastric, colorectal, or liver cancer; and theantisense S-oligonucleotide of RNF43 including those having thenucleotide sequence of SEQ ID NO: 31, suitably for colorectal, lung,gastric, or liver cancer. A suitable target sequence of siRNA has thenucleotide sequences selected from the group of SEQ ID NOs: 112, 113, or114. The target sequence of siRNA of CXADRL1 including those having thenucleotide sequence of SEQ ID NOs: 114 may be suitably used to treatgastric, colorectal, or liver cancer; and the target sequence of siRNAof RNF43 including those having the nucleotide sequence of SEQ ID NOs:112, or 113, suitably for colorectal, lung, gastric, or liver cancer.The pharmaceutical compositions may be also those comprising thecompounds selected by the present methods of screening for compounds fortreating cell proliferative diseases.

The course of action of the pharmaceutical composition is desirably toinhibit growth of the cancerous cells. The pharmaceutical compositionmay be applied to mammals including humans and domestic mammals.

The present invention further provides methods for treating a cellproliferative disease using the pharmaceutical composition provided bythe present invention.

In addition, the present invention provides method for treating orpreventing cancer, which method comprises the step of administering theCXADRL1, GCUD1, or RNF43 polypeptide. It is expected that anti-tumorimmunity be induced by the administration of the CXADRL1, GCUD1, orRNF43 polypeptide. Thus, the present invention also provides method forinducing anti-tumor immunity, which method comprises the step ofadministering the CXADRL1, GCUD1, or RNF43 polypeptide, as well aspharmaceutical composition for treating or preventing cancer comprisingthe CXADRL1, GCUD1, or RNF43 polypeptide.

It is to be understood that both the foregoing summary of the inventionand the following detailed description are of a preferred embodiment,and not restrictive of the invention or other alternate embodiments ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a to 1 d depict the expression of A5928 (CXADRL1) andC8121(GCUD1) in gastric cancers. FIG. 1 a depicts the relativeexpression ratios (cancer/non-cancer) of A5928 in primary 14 gastriccancers examined by cDNA microarray. Its expression was up-regulated(Cy3:Cy5 intensity ratio, >2.0) in 14 of the 14 gastric cancers thatpassed through the cutoff filter (both Cy3 and Cy5 signals greater than25,000). FIG. 1 b depicts the relative expression ratios(cancer/non-cancer) of C8121 in primary 12 gastric cancers examined bycDNA microarray. Its expression was up-regulated (Cy3:Cy5 intensityratio, >2.0) in 10 of the 12 gastric cancers that passed through thecutoff filter. FIG. 1 c depicts the expression of CXADRL1 analyzed bysemi-quantitative RT-PCR using 10 gastric cancer cases. FIG. 1 d depictsthe expression of GCUD1 analyzed by semi-quantitative RT-PCR using 9gastric cancer cases. Expression of GAPDH served as an internal controlfor both the expression analyses of CXADRL1 and GCUD1.

FIGS. 2 a and 2 b depict the expression of CXADRL1 in various humantissues and the predicted protein structure and protein motifs ofCXADRL1. FIG. 2 a is a photograph depicting expression of CXADRL1 invarious human tissues analyzed by multiple-tissue Northern-blotanalysis. FIG. 2 b depicts the predicted protein structure of CXADRL1.The CXADRL1 cDNA consists of 3,423 nucleotides with an ORF of 1,296nucleotides and is composed of 7 exons.

FIG. 3 a to 3 c depict the growth-promoting effect of CXADRL1. FIG. 3 ais a photograph depicting the result of colony formation assays ofNIH3T3 cells transfected with CXADRL1. FIG. 3 b depicts the expressionof exogeneous CXADRL1 in NIH3T3-CXADRL1 cells analyzed bysemi-quantitative RT-PCR. Expression of GAPDH served as an internalcontrol. #2, #5, #6, and #7 all indicate NIH3T3 cells transfected withCXADRL1. FIG. 3 c depicts the number of NIH3T3 cells. Growth ofNIH3T3-CXADRL1 cells was statistically higher than that of mock(NIH3T3-LacZ) cells in culture media containing 10% FBS (P<0.05).

FIG. 4 depicts the growth-inhibitory effect of antisenseS-oligonucleotides designated to suppress CXADRL1 in MKN-1 cells.CXADRL1-AS4 and CXADRL1-AS5 were demonstrated to suppress the growth ofMKN-1 cells.

FIG. 5A to 5C depict the growth suppressive effect of CXADRL1-siRNA onSt-4 cells. FIG. 5A presents photographs depicting the expression ofCXADRL1 and GAPDH (control) in St-4 cells transfected with mock orCXADRL1-siRNA#7. FIG. 5B depicts photographs depicting the result ofGiemsa's staining of viable cells treated with control-siRNA orCXADRL1-siRNA#7. FIG. 5C depicts the result of MTT assay on cellstransfected with control plasmid or plasmids expressing CXADRL1-siRNA7.

FIG. 6 depicts a photograph demonstrating the result of immunoblotanalysis of cells expressing exogeneous Flag-tagged CXADRL1 protein withanti-CXADRL1 antiserum or anti-Flag antibody.

FIG. 7 depicts the interaction between CXADRL1 and AIP1 examined byyeast two-hybrid system. FIG. 7 is a photograph depicting theinteraction of CXADRL1 with AIP1 examined by the two-hybrid system.

FIG. 8 depicts the peptide specific cytotoxicity of CTL line raised byCXADRL1-207 stimulation. The CTL line showed high cytotoxic activity ontarget cells (T2) pulsed with CXADRL1-207, whereas no significantcytotoxic activity was detected on the same target cells (T2) pulsedwithout peptides.

FIG. 9 depicts the cytotoxic activity of CXADRL1-207 CTL Clone onSNU475, MKN74, and SNU-C4. CXADRL1-207 CTL Clone showed high cytotoxicactivity on SNU475 that expresses both CXADRL1 and HLA-A*0201. On theother hand, CXADRL1-207 CTL Clone showed no significant cytotoxicactivity on MKN74, which expresses CXADRL1 but not HLA-A*0201.Furthermore, this CTL Clone did not show significant cytotoxic activityon SNU-C4, which expresses HLA-A*0201 but not CXADRL1.

FIG. 10 depicts the result of the cold target inhibition assay.CXADRL1-207 CTL Clone specifically recognizes CXADRL1-207 in anHLA-A*0201 restricted manner. SNU475 labeled with Na₂ ⁵¹CrO₄ wasprepared as a hot target, while CXADRL1-207 peptide-pulsed T2 (Peptide+)was used as a cold target (Inhibitors). E/T ratio was fixed to 20. Thecytotoxic activity on SNU475 was inhibited by the addition of T2 pulsedwith the identical peptide, while almost no inhibition by the additionof T2 without peptide pulse.

FIG. 11 depicts the result of the blocking assay showing the effect ofantibodies raised against HLA-Class I, HLA-Class II, CD4, and CD8 on thecytotoxic activity of CXADRL1-207 CTL Clone. CXADRL1-207 CTL Cloneshowed cytotoxic activity in HLA-Class I and CD8 restricted manner. Toexamine the characteristics of CTL clone raised with CXADRL1 peptide,antibodies against HLA-Class I, HLA-Class II, CD4, and CD8 were testedfor their ability to inhibit the cytotoxic activity. The horizontal axisreveals % inhibition of the cytotoxicity. The cytotoxicity of CTL cloneon SNU475 targets was significantly reduced when anti-class I and CD8antibodies were used. This result indicates that the CTL clonerecognizes the CXADRL1 derived peptide in a HLA-Class I and CD8dependant manner.

FIG. 12 depicts the cytotoxic activities of CTLs induced with anchormodified peptide CXADRL1-9V. The cytotoxic activities of CXADRL1-9Vinduced CTL line 5 (A) and CTL clone 69 (B) against peptide pulsed T2cells (HLA-A*0201 positive cell line) and tumor cell lines were examinedby 4 h ⁵¹Cr release assay. Both the CTL line 5 and CTL clone 69recognized not only CXADRL1-9V but also the parental peptide CXADRL1-9mer-207, equally or more sharply at a low E/T ratio in the CTL clone 69,and killed SNU475 cells expressing naturally processed wild-type peptideCXADRL1-9 mer-207 on the HLA-A*0201 molecule (C).

FIG. 13 is a photograph depicting the result of Northern-blot analysisof GCUD1 in various human tissues. The transcript of GCUD1 isapproximately 5.0-kb by size.

FIG. 14 shows a photograph depicting the subcellular localization ofGCUD1 observed by immunocytochemistry of cells transfected withpcDNA3.1myc/His-GCUD1. cMyc-tagged GCUD1 protein expressed from theplasmid localized in the cytoplasm.

FIG. 15 is a photograph showing the growth-promoting effect of GCUD1 onNIH3T3 cells examined by colony formation assays.

FIG. 16 depicts the growth-inhibitory effect of antisenseS-oligonucleotides designated to suppress GCUD1 on MKN-28 cells.GCUD1-AS5 and GCUD1-AS8 were revealed to suppress the growth of MKN-28cells.

FIG. 17 depicts a photograph showing the purification of recombinantGCUD1 protein.

FIG. 18 depicts a photograph demonstrating the result of immunoblotanalysis of cells expressing exogenous Flag-tagged GCUD1 protein withanti-GCUD1 antiserum or anti-Flag antibody.

FIG. 19 depicts the peptide specific cytotosicity of CTL line raised byGCUD1-196 or GCUD1-272 stimulation. The CTL line showed high cytotoxicactivity on target cells (T2) pulsed with GCUD1-196 or GCUD1-272,whereas no significant cytotoxic activity was detected on the sametarget cells (T2) pulsed without peptides.

FIG. 20 depicts the cytotoxic activity of GCUD1-196 CTL Clone on SNU475and MKN45. GCUD1-196 CTL Clone showed high cytotoxic activity on SNU475that expresses both GCUD1 and HLA-A*0201. On the other hand, GCUD1-196CTL Clone showed no significant cytotoxic activity on MKN45, whichexpresses GCUD1 but not HLA-A*0201.

FIG. 21 depicts the result of the cold target inhibition assay.GCUD1-196 CTL Clone specifically recognizes GCUD1-196 in an HLA-A*0201restricted manner. SNU475 labeled with Na₂ ⁵¹CrO₄ was prepared as a hottarget, while GCUD1-196 peptide-pulsed T2 (Peptide+) was used as a coldtarget (Inhibitors). E/T ratio was fixed to 20. The cytotoxic activityon SNU475 was inhibited by the addition of T2 pulsed with the identicalpeptide, while almost no inhibition was observed by the addition of T2without peptide pulse.

FIG. 22 depicts the result of the blocking assay showing the effect ofantibodies raised against HLA-Class I, HLA-Class II, CD4, and CD8 on thecytotoxic activity of GCUD1-196 CTL Clone. GCUD1-196 CTL Clone showedcytotoxic activity in HLA-Class I and CD8 restricted manner. To examinethe characteristics of CTL clone raised with GCUD1 peptide, antibodiesagainst HLA-Class I, HLA-Class II, CD4, and CD8 were tested for theirability to inhibit the cytotoxic activity. The horizontal axis reveals %inhibition of the cytotxicity. The cytotoxicity of CTL clone on SNU475targets was significantly reduced when anti-class I and CD8 antibodieswere used. This result indicates that the CTL clone recognizes the GCUD1derived peptide in a HLA Class I and CD8 dependent manner.

FIG. 23 depicts the cytotoxic activities of CTLs induced with anchormodified peptide GCUD1-9V. The cytotoxic activities of GCUD1-9V inducedCTL line 3 (A) and CTL clone 16 (B) against peptide pulsed T2 cells(HLA-A*0201 positive cell line) and tumor cell lines were examined by 4h ⁵¹Cr release assay. Both CTL line 3 and CTL clone 16 recognized notonly GCUD1-9V but also the parental peptide GCUD1-196, equally or moresharply at a low E/T ratio in CTL clone 16, and killed SNU475 cellsexpressing naturally processed wild-type peptide GCUD1-196 on theHLA-A*0201 molecule (C).

FIGS. 24 a and 24 b depict the expression of FLJ20315 in colon cancer.FIG. 24 a depicts the relative expression ratios (cancer/non-cancer) ofFLJ20315 in 11 primary colon cancer cases examined by cDNA microarray.Its expression was up-regulated (Cy3 :Cy5 intensity ratio, >2.0) in 10of the 11 colon cancer cases that passed through the cut-off filter(both Cy3 and Cy5 signals greater than 25,000). FIG. 24 b depicts theexpression of FLJ20315 analyzed by semi-quantitative RT-PCR usingadditional 18 colon cancer cases (T, tumor tissue; N, normal tissue).Expression of GAPDH served as an internal control.

FIG. 25 a depicts a photograph showing the result of fetal-tissueNorthern-blot analysis of RNF43 in various human fetal tissues. FIG. 25b depicts the predicted protein structure of RNF43.

FIGS. 26 a and 26 b show photographs depicting the subcellularlocalization of myc-tagged RNF43 protein. FIG. 26 a is a photographdepicting the result of Western-blot analysis of myc-tagged RNF43protein using extracts from COS7 cells transfected with eitherpcDNA3.1-myc/His-RNF43 or control plasmids (mock). FIG. 26 b presentsphotographs of the transfected cells that were stained with mouseanti-myc antibody and visualized by FITC-conjugated secondary antibody.Nuclei were counter-stained with DAPI.

FIG. 27 a to 27 c depicts the effect of RNF43 on cell growth. FIG. 27 ais a photograph depicting the result of colony formation assay of RNF43in NIH3T3 cells. FIG. 27 b presents photographs depicting the expressionof RNF43 in mock (COS7-pcDNA) and COS7-RNF43 cells that was establishedby the transfection of COS7 cells with pcDNA-RNF43. FIG. 27 c depictsthe result of comparison on cell growth between COS7-RNF43 cells stablyexpressing exogenous RNF43 and mock cells.

FIGS. 28 a and 28 b depict the growth-inhibitory effect of antisenseS-oligonucleotides designed to suppress RNF43. FIG. 28 a presentsphotographs depicting the expression of RNF43 in LoVo cells treated for12 h with either control (RNF43-S1) or antisense S-oligonucleotides(RNF43-AS1) analyzed by semi-quantitative RT-PCR. FIG. 28 b depicts thecell viability of LoVo cells after treatment with the control orantisense S-oligonucleotides measured by MTT assay. The MTT assay wascarried out in triplicate.

FIG. 29A to 29C depict the growth suppressive effect of RNF43-siRNAs.FIG. 29A presents photographs depicting the effect of RNF43-siRNAs onthe expression of RNF43. FIG. 29B presents photographs depicting theresult of Giemsa's staining of viable cells after the treatment withcontrol-siRNA or RNF43-siRNAs. FIG. 29C depicts the result of MTT assayon cells transfected with control plasmid or plasmids expressingRNF43-siRNAs. *, a significant difference (p<0.05) as determined by aFisher's protected least significant difference test.

FIGS. 30A and 30B depict the expression of tagged RNF43 protein. FIG.30A is a photograph depicting the result of Western-blot analysis ofFlag-tagged RNF43 protein secreted in the culture media of COS7 cellstransfected with pFLAG-5CMV-RNF43 (lane 2) or mock vector (lane 1). FIG.30B is a photograph depicting the result of Western-blot analysis ofMyc-tagged RNF43 protein secreted in the culture media of COS7 cellstransfected with pcDNA3.1-Myc/His-RNF43 (lane 2) or mock vector (lane1).

FIGS. 31A and 31B depict the growth promoting effect of conditionedmedia containing the Myc-tagged or Flag-tagged RNF43 protein. FIG. 31Apresents photographs depicting the morphology of NIH3T3 cells culturedin control media (1) or in conditioned media of COS7 cells transfectedwith mock vector (2), pcDNA3.1-Myc/His-RNF43 (3), or pFLAG-5CMV-RNF43(4). FIG. 31B depicts the number of NIH3T3 cells cultured in theindicated media described in FIG. 31A. Data are shown as means oftriplicate experiments for each group; bars, ±SE. *, significantdifference when compared with control, mock(p<0.05).

FIG. 32A to 32C depict the preparation of N-terminal (N1) and C-terminal(C1) recombinant protein of RNF43. FIG. 32A depicts the schematicstructure of the recombinant protein RNF43-N1 and -C1. FIG. 32B is aphotograph depicting the expression of Nus™-tagged RNF43-N1 protein inE. coli with (lane2) or without (lane 1) 0.2 mM of IPTG. FIG. 32C is aphotograph depicting the expression of Nus™-tagged RNF43-C1 protein inE. coli with (lane2) or without (lane 1) 1 mM of IPTG.

FIGS. 33A and 33B depict the interaction between RNF43 and NOTCH2examined by yeast two-hybrid system. FIG. 33A depicts the predictedstructure and the interacting region of NOTCH2. (a) shows the predictedfull-length structure of NOTCH2 protein, and (b) shows the predictedresponsible region for the interaction (ECD, Extracellular domain; TM,transmembrane domain; ICD, Intracellular domain). FIG. 33B is aphotograph depicting the interaction of RNF43 with NOTCH2 examined bythe two-hybrid system.

FIGS. 34A and 34B depict the interaction between RNF43 and STRINexamined by the yeast two-hybrid system. FIG. 34A depicts the predictedstructure and the interacting region of STRIN. (a) shows the predictedfull-length structure of STRIN protein, and (b) shows the predictedresponsible region for the interaction (RING, RING domain). FIG. 34B isa photograph depicting the interaction of RNF43 with STRIN examined bythe two-hybrid system.

FIG. 35 depicts the peptide specific cytotoxicity of CTL line raised byRNF43-721 stimulation. The CTL line showed high cytotoxic activity ontarget cells (TISI) pulsed with RNF43-721 (quadrilateral line), whereasno significant cytotoxic activity was detected on the same target cells(TISI) pulsed without peptides (triangular line). CTL line wasdemonstrated to have a peptide specific cytotoxicity.

FIG. 36 depicts the peptide specific cytotoxicity of CTL clones raisedby RNF43-721 stimulation. The cytotoxic activity of 13 RNF43-721 CTLclones on peptide-pulsed targets (TISI) was tested as described underthe item of “Materials and Methods”. The established RNF43-721 CTLclones had very potent cytotoxic activity on target cells (TISI) pulsedwith the peptides without showing any significant cytotoxic activity onthe same target cells (TISI) that were not pulsed with any peptides.

FIG. 37 depicts the cytotoxic activity of RNF43-721 CTL Clone 45 onHT29, WiDR and HCT116. RNF43-721 CTL Clone recognizes and lyses tumorcells that endogenously express RNF43 in an HLA restricted fashion.HT29, WiDR, and HCT116 all endogenously express RNF43, and RNF43-721 CTLClone 45 served as an effector cell. TISI was used as the target thatdoes not express RNF43. RNF43-721 CTL Clone 45 showed high cytotoxicactivity on HT29 (filled triangular line) and WiDR (diamond line) thatexpress both RNF43 and HLA-A24. On the other hand, RNF43-721 CTL Clone45 showed no significant cytotoxic activity on HCT116 (empty triangularline), which expresses RNF43 but not HLA-A24, and TISI (emptyquadrilateral line), which expresses HLA-A24 but not RNF43. Moreover,RNF43-721 CTL Clone 45 showed no cytotoxic activity on irrelevantpeptide pulsed TISI (filled quadrilateral dotted line) and SNU-C4(filled circle line) which expresses RNF43 but little HLA-A24.

FIG. 38 depicts the result of the cold target inhibition assay.RNF43-721 CTL Clone specifically recognizes RNF 43-721 in an HLA-A24restricted manner. HT29 labeled with Na₂ ⁵¹CrO₄ was prepared as a hottarget, while RNF43-721 peptide-pulsed TISI (Peptide+) was used as acold target (Inhibitors). E/T ratio was fixed to 20. The cytotoxicactivity on HT29 was inhibited by the addition of TISI pulsed with theidentical peptide (filled quadrilateral line), while almost noinhibition occurred by the addition of TISI without peptide pulsing(empty quadrilateral line).

FIG. 39 depicts the result of the blocking assay showing the effect ofantibodies raised against HLA-Class I, HLA-Class II, CD3, CD4, and CD8on the cytotoxic activity of RNF43-721 CTL Clone. RNF43-721 CTL Cloneshowed cytotoxic activity in HLA-Class I, CD3, and CD8 restrictedmanner. To examine the characteristics of CTL clone raised with RNF43peptide, antibodies against HLA-Class I, HLA-Class II, CD3, CD4, and CD8were tested for their ability to inhibit the cytotoxic activity. Thehorizontal axis reveals % inhibition of the cytotoxicity. Thecytotoxicity of CTL clone on WiDR targets was significantly reduced whenanti-class I, CD3, and CD8 antibodies were used. This result indicatesthat the CTL clone recognizes the RNF43 derived peptide in an HLA ClassI, CD3, and CD8 dependent manner.

FIGS. 40A and 40B depict the peptide specific cytotoxicity of the CTLlines raised with RNF43-11-9 (A) or RNF43-11-10 (B). These CTL linesshowed high cytotoxic activity on target cells (T2) pulsed withRNF43-11-9 or RNF43-11-10, whereas no significant cytotoxic activity wasobserved on the same target cells (T2) pulsed without peptides.

FIGS. 41A and 41B depict the peptide specific cytotoxicity of CTL clonesraised by RNF43-11-9 stimulation. Cytotoxic activity of 4 RNF43-11-9 CTLclones on peptide-pulsed targets (T2) was tested as described under theitem of “Materials and Methods”. The established RNF43-11-9 CTL cloneshad very potent cytotoxic activities on target cells (T2) pulsed withthe peptides without showing any significant cytotoxic activity on thesame target cells (T2) that were not pulsed with any peptides.

FIGS. 42A and 42B depict the cytotoxic activity of RNF43-5 CTL Clone 90and RNF43-17 CTL Clone 25 on HT29 and DLD-1. RNF43-5 CTL Clone 90 andRNF43-17 CTL Clone 25 recognize and lyse tumor cells that endogenouslyexpress RNF43 in an HLA restricted fashion. HT29 and DLD-1 allendogenously express RNF43, and RNF43-5 CTL Clone 90 and RNF43-17 CTLClone 25 served as an effector cell. T2 was used as the target that doesnot express RNF43. RNF43-5 CTL Clone 90 and RNF43-17 CTL Clone 25 showedhigh cytotoxic activity on DLD-1 that express both RNF43 and HLA-A*0201.On the other hand, RNF43-5 CTL Clone 90 and RNF43-17 CTL Clone 25 showedno significant cytotoxic activity on HT29, which expresses RNF43 but notHLA-A*0201.

FIG. 43 depicts the result of cold target inhibition assay. RNF43 CTLClone specifically recognizes RNF 43 in a HLA-A2 restricted manner.HCT116 labeled with Na₂ ⁵¹CrO₄ was prepared as a hot target, while RNF43peptide-pulsed T2 (Peptide+) was used as a cold target (Inhibitors). E/Tratio was fixed to 20. The cytotoxic activity on HCT116 was inhibited bythe addition of T2 pulsed with the identical peptide, while almost noinhibition was observed by the addition of TISI without peptide pulse.

DETAILED DESCRIPTION OF THE INVENTION

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

The present application identifies novel human genes CXADRL1 and GCUD1whose expression is markedly elevated in gastric cancer compared tocorresponding non-cancerous tissues. The CXADRL1 cDNA consists of 3423nucleotides that contain an open reading frame of 1296 nucleotides asset forth in SEQ ID NO: 1. The open reading frame encodes a putative431-amino acid protein. CXADRL1 associates with atripin-1-interactingprotein 1 (AIP1). AIP1 is a protein that associates with atripin-1, agene responsible for a hereditary disease, dentatorubral-pallidoluysianatrophy. AIP1 encodes a deduced 1455-amino acid protein containingguanylate kinase-like domain, two WW domains and five PDZ domains. Themouse homolog of AIP1 was shown to interact with activin type IIA.However, the function of AIP1 remains to be resolved. The predictedamino acid sequence showed an identity of about 37% to coxsackie andadenovirus receptor (CXADR). Therefore this protein was dubbed coxsackieand adenovirus receptor like 1 (CXADRL1). On the other hand, the GCUD1cDNA consists of 4987 nucleotides that contain an open reading frame of1245 nucleotides as set forth in SEQ ID NO: 3. The open reading frameencodes a putative 414-amino acid protein. Since the expression of theprotein was up-regulated in gastric cancer, the protein was dubbed GCUD1(up-regulated in gastric cancer).

Furthermore, the present invention encompasses novel human gene RNF43whose expression is markedly elevated in colorectal cancer compared tocorresponding non-cancerous tissue. The RNF43 cDNA consists of 5345nucleotides that contain an open reading frame of 2352 nucleotides asset forth in SEQ ID NO: 5. The open reading frame encodes a putative783-amino acid protein. RNF43 associates with NOTCH2 and STRIN. NOTCH2is reported as a large transmembrane receptor protein that is acomponent of an evolutionarily conserved intercellular signalingmechanism. NOTCH2 is a protein member of the Notch signaling pathway andis reported to be involved in glomerulogenesis in the kidney anddevelopment of heart and eye vasculature. Furthermore, threeDelta/Serrate/Lag-2 (DSL) proteins, Delta1, Jaggaed1, and Jaggaed2, arereported as functional ligands for NOTCH2. STRIN encodes a putativeprotein that shares 79% identity with mouse Trif. The function of STRINor Trif remains to be clarified.

Consistently, exogenous expression of CXADRL1, GCUD1, or RNF43 intocells conferred increased cell growth, while suppression of itsexpression with antisense S-oligonucleotides or small interfering RNA(siRNA) resulted in significant growth-inhibition of cancerous cells.These findings suggest that CXADRL1, GCUD1, and RNF43 render oncogenicactivities to cancer cells, and that inhibition of the activity of theseproteins could be a promising strategy for the treatment of cancer.

The present invention encompasses novel human gene CXADRL1, including apolynucleotide sequence as described in SEQ ID NO: 1, as well asdegenerates and mutants thereof, to the extent that they encode aCXADRL1 protein, including the amino acid sequence set forth in SEQ IDNO: 2 or its functional equivalent. Examples of polypeptidesfunctionally equivalent to CXADRL1 include, for example, homologousproteins of other organisms corresponding to the human CXADRL1 protein,as well as mutants of human CXADRL1 proteins.

The present invention also encompasses novel human gene GCUD1, includinga polynucleotide sequence as described in SEQ ID NO: 3, as well asdegenerates and mutants thereof, to the extent that they encode a GCUD1protein, including the amino acid sequence set forth in SEQ ID NO: 4 orits functional equivalent. Examples of polypeptides functionallyequivalent to GCUD1 include, for example, homologous proteins of otherorganisms corresponding to the human GCUD1 protein, as well as mutantsof human GCUD1 proteins.

Furthermore, the present invention encompasses novel human gene RNF43,including a polynucleotide sequence as described in SEQ ID NO: 5, aswell as degenerates and mutants thereof, to the extent that they encodea RNF43 protein, including the amino acid sequence set forth in SEQ IDNO: 6 or its functional equivalent. Examples of polypeptidesfunctionally equivalent to RNF43 include, for example, homologousproteins of other organisms corresponding to the human RNF43 protein, aswell as mutants of human RNF43 proteins.

In the present invention, the phrase “functionally equivalent” meansthat the subject polypeptide has activities to promote cellproliferation like CXADRL1, GCUD1, or RNF43 protein and to conferoncogenic activity to cancer cells. Whether the subject polypeptide hasa cell proliferation activity or not can be judged by introducing theDNA encoding the subject polypeptide into a cell expressing therespective polypeptide, and detecting promotion of proliferation of thecells or increase in colony forming activity. Such cells include, forexample, NIH3T3 cells for CXADRL1 and GCUD1; and NIH3T3 cells, SW480cells, and COS7 cells for RNF43. Alternatively, whether the subjectpolypeptide is functionally equivalent to CXADRL1 may be judged bydetecting its binding ability to AIP1. Furthermore, whether the subjectpolypeptide is functionally equivalent to RNF43 may be judged bydetecting its binding ability to NOTCH2 or STRIN.

Methods for preparing polypeptides functionally equivalent to a givenprotein are well known by a person skilled in the art and include knownmethods of introducing mutations into the protein. For example, oneskilled in the art can prepare polypeptides functionally equivalent tothe human CXADRL1, GCUD 1, or RNF43 protein by introducing anappropriate mutation in the amino acid sequence of either of theseproteins by site-directed mutagenesis (Hashimoto-Gotoh et al., Gene152:271-5 (1995); Zoller and Smith, Methods Enzymol 100: 468-500 (1983);Kramer et al., Nucleic Acids Res. 12:9441-9456 (1984); Kramer and Fritz,Methods Enzymol 154: 350-67 (1987); Kunkel, Proc Natl Acad Sci USA 82:488-92 (1985); Kunkel, Methods Enzymol 85: 2763-6 (1988)). Amino acidmutations can occur in nature, too. The polypeptide of the presentinvention includes proteins having the amino acid sequences of the humanCXADRL1, GCUD1, or RNF43 protein in which one or more amino acids aremutated, provided the resulting mutated polypeptides are functionallyequivalent to the human CXADRL1, GCUD1, or RNF43 protein. The number ofamino acids to be mutated in such a mutant is generally 10 amino acidsor less, preferably 6 amino acids or less, and more preferably 3 aminoacids or less.

Mutated or modified proteins, proteins having amino acid sequencesmodified by substituting, deleting, inserting, and/or adding one or moreamino acid residues of a certain amino acid sequence, have been known toretain the original biological activity (Mark et al., Proc Natl Acad SciUSA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500(1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13(1982)).

The amino acid residue to be mutated is preferably mutated into adifferent amino acid in which the properties of the amino acidside-chain are conserved (a process known as conservative amino acidsubstitution). Examples of properties of amino acid side chains arehydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic aminoacids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having thefollowing functional groups or characteristics in common: an aliphaticside-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain(S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acidand amide containing side-chain (D, N, E, Q); a base containingside-chain (R, K, H); and an aromatic containing side-chain (H, F, Y,W). Note, the parenthetic letters indicate the one-letter codes of aminoacids.

An example of a polypeptide to which one or more amino acids residuesare added to the amino acid sequence of human CXADRL1, GCUD1, or RNF43protein is a fusion protein containing the human CXADRL1, GCUD1, orRNF43 protein. Fusion proteins are, fusions of the human CXADRL1, GCUD1,or RNF43 protein and other peptides or proteins, and are included in thepresent invention. Fusion proteins can be made by techniques well knownto a person skilled in the art, such as by linking the DNA encoding thehuman CXADRL1, GCUD1, or RNF43 protein of the invention with DNAencoding other peptides or proteins, so that the frames match, insertingthe fusion DNA into an expression vector and expressing it in a host.There is no restriction as to the peptides or proteins fused to theprotein of the present invention.

Known peptides that can be used as peptides that are fused to theprotein of the present invention include, for example, FLAG (Hopp etal., Biotechnology 6: 1204-10 (1988)), 6xHis containing six His(histidine) residues, 10× His, Influenza agglutinin (HA), human c-mycfragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag,SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment, and the like. Examples of proteins that may be fused to aprotein of the invention include GST (glutathione-S-transferase),Influenza agglutinin (HA), immunoglobulin constant region,β-galactosidase, MBP (maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNA,encoding the fusion peptides or proteins discussed above, with the DNAencoding the polypeptide of the present invention and expressing thefused DNA prepared.

An alternative method known in the art to isolate functionallyequivalent polypeptides is, for example, the method using ahybridization technique (Sambrook et al., Molecular Cloning 2nd ed.9.47-9.58, Cold Spring Harbor Lab. Press (1989)). One skilled in the artcan readily isolate a DNA having high homology with a whole or part ofthe DNA sequence encoding the human CXADRL1, GCUD1, or RNF43 protein(i.e., SEQ ID NO: 1, 3, or 5), and isolate functionally equivalentpolypeptides to the human CXADRL1, GCUD1, or RNF43 protein from theisolated DNA. The polypeptides of the present invention include thosethat are encoded by DNA that hybridize with a whole or part of the DNAsequence encoding the human CXADRL1, GCUD1, or RNF43 protein and arefunctionally equivalent to the human CXADRL1, GCUD1, or RNF43 protein.These polypeptides include mammal homologues corresponding to theprotein derived from human (for example, a polypeptide encoded by amonkey, rat, rabbit or bovine gene). In isolating a cDNA highlyhomologous to the DNA encoding the human CXADRL1 protein from animals,it is particularly preferable to use tissues from testis or ovary.Alternatively, in isolating a cDNA highly homologous to the DNA encodingthe human GCUD1 from animals, it is particularly preferable to usetissues from testis, ovary, or brain. Further, in isolating a cDNAhighly homologous to the DNA encoding the human RNF43 protein fromanimals, it is particularly preferable to use tissue from fetal lung orfetal kidney.

The condition of hybridization for isolating a DNA encoding apolypeptide functionally equivalent to the human CXADRL1, GCUD1, orRNF43 protein can be routinely selected by a person skilled in the art.For example, hybridization may be performed by conductingprehybridization at 68° C. for 30 min or longer using “Rapid-hyb buffer”(Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68° C.for 1 hour or longer. The following washing step can be conducted, forexample, in a low stringent condition. A low stringent condition is, forexample, 42° C., 2×SSC, 0.1% SDS, or preferably 50° C., 2×SSC, 0.1% SDS.More preferably, high stringent conditions are used. A high stringentcondition is, for example, washing 3 times in 2×SSC, 0.01% SDS at roomtemperature for 20 min, then washing 3 times in 1×SSC, 0.1% SDS at 37°C. for 20 min, and washing twice in 1×SSC, 0.1% SDS at 50° C. for 20min. However, several factors in addition to temperature and saltconcentration, such as length of the probe and GC content of the probe,can influence the stringency of hybridization and one skilled in the artcan suitably select the factors to achieve the requisite stringency.

In place of hybridization, a gene amplification method, for example, thepolymerase chain reaction (PCR) method, can be utilized to isolate a DNAencoding a polypeptide functionally equivalent to the human CXADRL1,GCUD1, or RNF43 protein, using a primer synthesized based on thesequence information of the protein encoding DNA (SEQ ID NO: 1, 3, or5).

Polypeptides that are functionally equivalent to the human CXADRL1,GCUD1, or RNF43 protein encoded by the DNA isolated through the abovehybridization techniques or gene amplification techniques, normally havea high homology to the amino acid sequence of the human CXADRL1, GCUD1,or RNF43 protein. “High homology” typically refers to a homology of 40%or higher, preferably 60% or higher, more preferably 80% or higher, evenmore preferably 95% or higher. The homology of a polypeptide can bedetermined by following the algorithm in “Wilbur and Lipman, Proc NatlAcad Sci USA 80: 726-30 (1983)”.

A polypeptide of the present invention may have variations in amino acidsequence, molecular weight, isoelectric point, the presence or absenceof sugar chains, or form, depending on the cell or host used to produceit or the purification method utilized. Nevertheless, so long as it hasa function equivalent to that of the human CXADRL1, GCUD1, or RNF43protein of the present invention, it is within the scope of the presentinvention.

The polypeptides of the present invention can be prepared as recombinantproteins or natural proteins, by methods well known to those skilled inthe art. A recombinant protein can be prepared by inserting a DNA, whichencodes the polypeptide of the present invention (for example, the DNAcomprising the nucleotide sequence of SEQ ID NO: 1, 3, or 5), into anappropriate expression vector, introducing the vector into anappropriate host cell, obtaining the extract, and purifying thepolypeptide by subjecting the extract to chromatography, for example,ion exchange chromatography, reverse phase chromatography, gelfiltration, or affinity chromatography utilizing a column to whichantibodies against the protein of the present invention is fixed, or bycombining more than one of the aforementioned columns.

Also when the polypeptide of the present invention is expressed withinhost cells (for example, animal cells and E. coli) as a fusion proteinwith glutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column.Alternatively, when the polypeptide of the present invention isexpressed as a protein tagged with c-myc, multiple histidines, or FLAG,it can be detected and purified using antibodies to c-myc, His, or FLAG,respectively.

After purifying the fusion protein, it is also possible to excluderegions other than the objective polypeptide by cutting with thrombin orfactor-Xa as required.

A natural protein can be isolated by methods known to a person skilledin the art, for example, by contacting the affinity column, in whichantibodies binding to the CXADRL1, GCUD1, or RNF43 protein describedbelow are bound, with the extract of tissues or cells expressing thepolypeptide of the present invention. The antibodies can be polyclonalantibodies or monoclonal antibodies.

The present invention also encompasses partial peptides of thepolypeptide of the present invention. The partial peptide has an aminoacid sequence specific to the polypeptide of the present invention andconsists of at least 7 amino acids, preferably 8 amino acids or more,and more preferably 9 amino acids or more. The partial peptide can beused, for example, for preparing antibodies against the polypeptide ofthe present invention, screening for a compound that binds to thepolypeptide of the present invention, screening for accelerators orinhibitors of the polypeptide of the present invention, and as atumor-associated antigen (TAA).

A partial peptide of the invention can be produced by geneticengineering, by known methods of peptide synthesis, or by digesting thepolypeptide of the invention with an appropriate peptidase. For peptidesynthesis, for example, solid phase synthesis or liquid phase synthesismay be used.

Furthermore, the present invention provides polynucleotides encoding thepolypeptide of the present invention. The polynucleotides of the presentinvention can be used for the in vivo or in vitro production of thepolypeptide of the present invention as described above, or can beapplied to gene therapy for diseases attributed to genetic abnormalityin the gene encoding the protein of the present invention. Any form ofthe polynucleotide of the present invention can be used so long as itencodes the polypeptide of the present invention, including mRNA, RNA,cDNA, genomic DNA, chemically synthesized polynucleotides. Thepolynucleotide of the present invention include a DNA comprising a givennucleotide sequences as well as its degenerate sequences, so long as theresulting DNA encodes a polypeptide of the present invention.

The polynucleotide of the present invention can be prepared by methodsknown to a person skilled in the art. For example, the polynucleotide ofthe present invention can be prepared by: preparing a cDNA library fromcells which express the polypeptide of the present invention, andconducting hybridization using a partial sequence of the DNA of thepresent invention (for example, SEQ ID NO: 1, 3, or 5) as a probe. AcDNA library can be prepared, for example, by the method described inSambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press(1989); alternatively, commercially available cDNA libraries may beused. A cDNA library can be also prepared by: extracting RNAs from cellsexpressing the polypeptide of the present invention, synthesizing oligoDNAs based on the sequence of the DNA of the present invention (forexample, SEQ ID NO: 1, 3, or 5), conducting PCR using the oligo DNAs asprimers, and amplifying cDNAs encoding the protein of the presentinvention.

In addition, by sequencing the nucleotides of the obtained cDNA, thetranslation region encoded by the cDNA can be routinely determined, andthe amino acid sequence of the polypeptide of the present invention canbe easily obtained. Moreover, by screening the genomic DNA library usingthe obtained cDNA or parts thereof as a probe, the genomic DNA can beisolated.

More specifically, mRNAs may first be prepared from a cell, tissue, ororgan (e.g., testis or ovary for CXADRL1; testis, ovary, or brain forGCUD1; and fetal lung, or fetal kidney for RNF43) in which the objectpolypeptide of the invention is expressed. Known methods can be used toisolate mRNAs; for instance, total RNA may be prepared by guanidineultracentrifugation (Chirgwin et al., Biochemistry 18:5294-9 (1979)) orAGPC method (Chomczynski and Sacchi, Anal Biochem 162:156-9 (1987)). Inaddition, mRNA may be purified from total RNA using mRNA PurificationKit (Pharmacia) and such or, alternatively, mRNA may be directlypurified by QuickPrep mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized using a commercially availablekit, such as the AMV Reverse Transcriptase First-strand cDNA SynthesisKit (Seikagaku Kogyo). Alternatively, cDNA may be synthesized andamplified following the 5′-RACE method (Frohman et al., Proc Natl AcadSci USA 85: 8998-9002 (1988); Belyavsky et al., Nucleic Acids Res 17:2919-32 (1989)), which uses a primer and such, described herein, the5′-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction(PCR).

A desired DNA fragment is prepared from the PCR products and ligatedwith a vector DNA. The recombinant vectors are used to transform E. coliand such, and a desired recombinant vector is prepared from a selectedcolony. The nucleotide sequence of the desired DNA can be verified byconventional methods, such as dideoxynucleotide chain termination.

The nucleotide sequence of a polynucleotide of the invention may bedesigned to be expressed more efficiently by taking into account thefrequency of codon usage in the host to be used for expression (Granthamet al., Nucleic Acids Res 9: 43-74 (1981)). The sequence of thepolynucleotide of the present invention may be altered by a commerciallyavailable kit or a conventional method. For instance, the sequence maybe altered by digestion with restriction enzymes, insertion of asynthetic oligonucleotide or an appropriate polynucleotide fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG).

Specifically, the polynucleotide of the present invention encompassesthe DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3, or 5.

Furthermore, the present invention provides a polynucleotide thathybridizes under stringent conditions with a polynucleotide having anucleotide sequence of SEQ ID NO: 1, 3, or 5, and encodes a polypeptidefunctionally equivalent to the CXADRL1, GCUD1, or RNF43 protein of theinvention described above. One skilled in the art may appropriatelychoose a stringent condition. For example, low stringent condition canbe used. More preferably, high stringent condition can be used. Theseconditions are the same as those described above. The hybridizing DNAabove is preferably a cDNA or a chromosomal DNA.

The present invention also provides a vector into which a polynucleotideof the present invention is inserted. A vector of the present inventionis useful to keep a polynucleotide, especially a DNA, of the presentinvention in host cell, to express the polypeptide of the presentinvention, or to administer the polynucleotide of the present inventionfor gene therapy.

When E. coli is a host cell and the vector is amplified and produced ina large amount in E. coli (e.g., JM109, DH5α, HB101, or XL1Blue), thevector should have “ori” to be amplified in E. coli and a marker genefor selecting transformed E. coli (e.g., a drug-resistance gene selectedby a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol,or the like). For example, M13-series vectors, pUC-series vectors,pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T,pDIRECT, and pT7 can also be used for subcloning and extracting cDNA aswell as the vectors described above. When a vector is used to producethe protein of the present invention, an expression vector isparticularly useful. For example, an expression vector to be expressedin E. coli should have the above characteristics to be amplified in E.coli. When E. coli, such as JM109, DH5α, HB101, or XL1 Blue, are used asa host cell, the vector should have a promoter, for example, lacZpromoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7(1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7promoter, or the like, that can efficiently express the desired gene inE. coli. In that respect, pGEX-5X-1 (Pharmacia), “QIAexpress system”(Qiagen), pEGFP, and pET (in this case, the host is preferably BL21which expresses T7 RNA polymerase), for example, can be used instead ofthe above vectors. Additionally, the vector may also contain a signalsequence for polypeptide secretion. An exemplary signal sequence thatdirects the polypeptide to be secreted to the periplasm of the E. coliis the pelB signal sequence (Lei et al., J Bacteriol 169: 4379-83(1987)). Means for introducing of the vectors into the target host cellsinclude, for example, the calcium chloride method, and theelectroporation method.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Mizushima etal., Nucleic Acids Res 18(17): 5322 (1990)), pEF, pCDM8), expressionvectors derived from insect cells (for example, “Bac-to-BAC baculovirusexpression system” (GIBCO BRL), pBacPAK8), expression vectors derivedfrom plants (e.g., pMH1, pMH2), expression vectors derived from animalviruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived fromretroviruses (e.g., pZIpneo), expression vector derived from yeast(e.g., “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01), andexpression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50)can be used for producing the polypeptide of the present invention.

In order to express the vector in animal cells, such as CHO, COS, orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al., Nature277: 108-14 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushimaet al., Nucleic Acids Res 18: 5322 (1990)), the CMV promoter, and thelike; and preferably a marker gene for selecting transformants (forexample, a drug resistance gene selected by a drug (e.g., neomycin,G418)). Examples of known vectors with these characteristics include,for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

In addition, methods may be used to stably express a gene and, at thesame time, to amplify the copy number of the gene in cells. For example,a vector comprising the complementary DHFR gene (e.g., pCHO I) may beintroduced into CHO cells in which the nucleic acid synthesizing pathwayis deleted, and then amplified by methotrexate (MTX). Furthermore, incase of transient expression of a gene, the method wherein a vectorcomprising a replication origin of SV40 (pcD, etc.) is transformed intoCOS cells comprising the SV40 T antigen expressing gene on thechromosome can be used.

A polypeptide of the present invention obtained as above may be isolatedfrom inside or outside (such as medium) of host cells, and purified as asubstantially pure homogeneous polypeptide. The phrase “substantiallypure” as used herein in reference to a given polypeptide means that thepolypeptide is substantially free from other biological macromolecules.The substantially pure polypeptide is at least 75% (e.g., at least 80,85, 95, or 99%) pure by dry weight. Purity can be measured by anyappropriate standard method, for example, by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis. The method forpolypeptide isolation and purification is not limited to any specificmethod; in fact, any standard method may be used.

For instance, column chromatography, filter, ultrafiltration, saltprecipitation, solvent precipitation, solvent extraction, distillation,immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectricpoint electrophoresis, dialysis, and recrystallization may beappropriately selected and combined to isolate and purify thepolypeptide.

Examples of chromatography include, for example, affinitychromatography, ion-exchange chromatography, hydrophobic chromatography,gel filtration, reverse phase chromatography, adsorption chromatography,and such (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed. Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). These chromatographies may be performedby liquid chromatography, such as HPLC and FPLC. Thus, the presentinvention provides highly purified polypeptides prepared by the abovemethods.

A polypeptide of the present invention may be optionally modified orpartially deleted by treating it with an appropriate proteinmodification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, glucosidase, and soon.

The present invention provides an antibody that binds to the polypeptideof the invention. The antibody of the invention can be used in any form,such as monoclonal or polyclonal antibodies, and includes antiserumobtained by immunizing an animal such as rabbit with the polypeptide ofthe invention, all classes of polyclonal and monoclonal antibodies,human antibodies, and humanized antibodies produced by geneticrecombination.

A polypeptide of the invention used as an antigen to obtain an antibodymay be derived from any animal species, but preferably is derived from amammal such as human, mouse, or rat, more preferably from human. Ahuman-derived polypeptide may be obtained from the nucleotide or aminoacid sequences disclosed herein.

According to the present invention, the polypeptide to be used as animmunization antigen may be a complete protein or a partial peptide ofthe protein. A partial peptide may comprise, for example, the amino(N)-terminal or carboxy (C)-terminal fragment of a polypeptide of thepresent invention. More specifically, a polypeptide of CXADRL1encompassing the codons from 235 to 276, from 493 to 537, or from 70 to111 can be used as partial peptides for producing antibodies againstCXADRL1 of the present invention.

Alternatively, for the production of antibodies against the polypeptideof the present invention, peptides comprising any one of following aminoacid sequences may be used:

-   -   RNF43; SEQ ID No: 80, 97, or 108;    -   CXADRL1 ; SEQ ID No: 124; and    -   GCUD1; SEQ ID No: 164.        Herein, an antibody is defined as a protein that reacts with        either the full-length or a fragment of a polypeptide of the        present invention.

A gene encoding a polypeptide of the invention or its fragment may beinserted into a known expression vector, which is then used to transforma host cell as described herein. The desired polypeptide or its fragmentmay be recovered from the outside or inside of host cells by anystandard method, and may subsequently be used as an antigen.Alternatively, whole cells expressing the polypeptide or their lysates,or a chemically synthesized polypeptide may be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha, or Primates areused. Animals of Rodentia include, for example, mouse, rat, and hamster.Animals of Lagomorpha include, for example, rabbit. Animals of Primatesinclude, for example, a monkey of Catarrhini (old world monkey) such asMacaca fascicularis, rhesus monkey, sacred baboon, and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for immunization of mammals. More specifically, antigensmay be diluted and suspended in an appropriate amount of phosphatebuffered saline (PBS), physiological saline, etc. If desired, theantigen suspension may be mixed with an appropriate amount of a standardadjuvant, such as Freund's complete adjuvant, made into emulsion, andthen administered to mammalian animals. Preferably, it is followed byseveral administrations of antigen mixed with an appropriately amount ofFreund's incomplete adjuvant every 4 to 21 days. An appropriate carriermay also be used for immunization. After immunization as above, serum isexamined by a standard method for an increase in the amount of desiredantibodies.

Polyclonal antibodies against the polypeptides of the present inventionmay be prepared by collecting blood from the immunized mammal examinedfor the increase of desired antibodies in the serum, and by separatingserum from the blood by any conventional method. Polyclonal antibodiesinclude serum containing the polyclonal antibodies, as well as thefraction containing the polyclonal antibodies isolated from the serum.Immunoglobulin G or M can be prepared from a fraction which recognizesonly the polypeptide of the present invention using, for example, anaffinity column coupled with the polypeptide of the present invention,and further purifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammals, andmore preferably myeloma cells having an acquired property that enablesselection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre andMilstein, Methods Enzymol 73: 3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all other cells, withthe exception of the desired hybridoma (non-fused cells), to die. Then,the standard limiting dilution is performed to screen and clone ahybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal isimmunized with an antigen for preparing hybridoma, human lymphocytessuch as those infected by EB virus may be immunized with a polypeptide,polypeptide expressing cells, or their lysates in vitro. Then, theimmunized lymphocytes are fused with human-derived myeloma cells thatare capable of indefinitely dividing, such as U266, to yield a hybridomaproducing a desired human antibody that is able to bind to thepolypeptide can be obtained (Unexamined Published Japanese PatentApplication No. (JP-A) Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are extracted. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which the polypeptide of thepresent invention is coupled. The antibody of the present invention canbe used not only for purification and detection of the polypeptide ofthe present invention, but also is a candidate for agonists andantagonists of the polypeptide of the present invention. In addition,this antibody can be applied to antibody treatment for diseases relatedto the polypeptide of the present invention. When the obtained antibodyis administered to the human body (antibody treatment), a human antibodyor a humanized antibody is preferable for reducing immunogenicity.

For example, transgenic animals having a repertory of human antibodygenes may be immunized with an antigen selected from a polypeptide,polypeptide expressing cells, or their lysates. Antibody producing cellsare then collected from the animals and fused with myeloma cells toobtain hybridoma, from which human antibodies against the polypeptidecan be prepared (see WO92-03918, WO93-02227, WO94-02602, WO94-25585,WO96-33735, and WO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck andLarrick, Therapeutic Monoclonal Antibodies, published in the UnitedKingdom by MacMillan Publishers LTD (1990)). For example, a DNA encodingan antibody may be cloned from an immune cell, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, so long as it binds to one or more ofthe polypeptides of the invention. For instance, the antibody fragmentmay be Fab, F(ab′)₂, Fv, or single chain Fv (scFv), in which Fvfragments from H and L chains are ligated by an appropriate linker(Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). Morespecifically, an antibody fragment may be generated by treating anantibody with an enzyme, such as papain or pepsin. Alternatively, a geneencoding the antibody fragment may be constructed, inserted into anexpression vector, and expressed in an appropriate host cell (see, forexample, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz,Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, MethodsEnzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986);Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker,Trends Biotechnol 9: 132-7 (1991)).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention furtherprovides such modified antibodies. The modified antibody can be obtainedby chemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, between a variable region derived from nonhumanantibody and the constant region derived from human antibody, or as ahumanized antibody, comprising the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) derivedfrom human antibody, and the constant region. Such antibodies can beprepared using known technology.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing, and others (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988)), but are not limited thereto. A protein A column andprotein G column can be used as the affinity column. Exemplary protein Acolumns to be used include, for example, Hyper D, POROS, and SepharoseF.F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography, andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). The chromatographic procedures can becarried out by liquid-phase chromatography, such as HPLC, and FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, a polypeptide of the invention isapplied to the plate, and then a sample containing a desired antibody,such as culture supernatant of antibody producing cells or purifiedantibodies, is applied. Then, a secondary antibody that recognizes theprimary antibody and is labeled with an enzyme, such as alkalinephosphatase, is applied, and the plate is incubated. Next, afterwashing, an enzyme substrate, such as p-nitrophenyl phosphate, is addedto the plate, and the absorbance is measured to evaluate the antigenbinding activity of the sample. A fragment of the polypeptide, such as aC-terminal or N-terminal fragment, may be used as the antigen toevaluate the binding activity of the antibody. BIAcore (Pharmacia) maybe used to evaluate the activity of the antibody according to thepresent invention.

The above methods allow for the detection or measurement of thepolypeptide of the invention, by exposing the antibody of the inventionto a sample assumed to contain the polypeptide of the invention, anddetecting or measuring the immune complex formed by the antibody and thepolypeptide.

Because the method of detection or measurement of the polypeptideaccording to the invention can specifically detect or measure apolypeptide, the method may be useful in a variety of experiments inwhich the polypeptide is used.

The present invention also provides a polynucleotide which hybridizeswith the polynucleotide encoding human CXADRL1, GCUD1, or RNF43 protein(SEQ ID NO: 1, 3, or 5) or the complementary strand thereof, and whichcomprises at least 15 nucleotides. The polynucleotide of the presentinvention is preferably a polynucleotide which specifically hybridizeswith the DNA encoding the polypeptide of the present invention. Thephrase “specifically hybridize” as used herein, means thatcross-hybridization does not occur significantly with DNA encoding otherproteins, under the usual hybridizing conditions, preferably understringent hybridizing conditions. Such polynucleotides include, probes,primers, nucleotides and nucleotide derivatives (for example, antisenseoligonucleotides and ribozymes), which specifically hybridize with DNAencoding the polypeptide of the invention or its complementary strand.Moreover, such polynucleotide can be utilized for the preparation of DNAchip.

The present invention includes an antisense oligonucleotide thathybridizes with any site within the nucleotide sequence of SEQ ID NO: 1,3, or 5. This antisense oligonucleotide is preferably against at least15 continuous nucleotides of the nucleotide sequence of SEQ ID NO: 1, 3,or 5. The above-mentioned antisense oligonucleotide, which contains aninitiation codon in the above-mentioned at least 15 continuousnucleotides, is even more preferred. More specifically, such antisenseoligonucleotides include those comprising the nucleotide sequence of SEQID NO: 23 or 25 for suppressing the expression of CXADRL1; SEQ ID NO:27, or 29 for GCUD1; and SEQ ID NO: 31 for RNF43.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsinclude lower alkyl phosphonate modifications, such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioatemodifications and phosphoroamidate modifications.

The phrase “antisense oligonucleotides” as used herein means, not onlythose in which the nucleotides corresponding to those constituting aspecified region of a DNA or mRNA are entirely complementary, but alsothose having a mismatch of one or more nucleotides, as long as the DNAor mRNA and the antisense oligonucleotide can specifically hybridizewith the nucleotide sequence of SEQ ID NO: 1, 3, or 5.

Such polynucleotides are contained as those having, in the “at least 15continuous nucleotide sequence region”, a homology of at least 70% orhigher, preferably at 80% or higher, more preferably 90% or higher, evenmore preferably 95% or higher. The algorithm stated herein can be usedto determine the homology. Such polynucleotides are useful as probes forthe isolation or detection of DNA encoding the polypeptide of theinvention as stated in a later example or as a primer used foramplifications.

The antisense oligonucleotide derivatives of the present invention actupon cells producing the polypeptide of the invention by binding to theDNA or mRNA encoding the polypeptide, inhibiting its transcription ortranslation, promoting the degradation of the mRNA, and inhibiting theexpression of the polypeptide of the invention, thereby resulting in theinhibition of the polypeptide's function.

An antisense oligonucleotide derivative of the present invention can bemade into an external preparation, such as a liniment or a poultice, bymixing with a suitable base material which is inactive against thederivatives.

Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops, and freeze-dried agents by adding excipients, isotonicagents, solubilizers, stabilizers, preservatives, pain-killers, andsuch. These can be prepared following usual methods.

The antisense oligonucleotide derivative is given to the patient bydirectly applying onto the ailing site or by injecting into a bloodvessel so that it will reach the site of ailment. An antisense-mountingmedium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L-lysine, lipid,cholesterol, lipofectin, or derivatives of these.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The present invention also includes small interfering RNAs (siRNA)comprising a combination of a sense strand nucleic acid and an antisensestrand nucleic acid of the nucleotide sequence of SEQ ID NO: 1, 3, or 5.

The term “siRNA” refers to a double-stranded RNA molecule which preventstranslation of a target mRNA. Standard techniques are used forintroducing siRNA into cells, including those wherein DNA is used as thetemplate to transcribe RNA. The siRNA comprises a sense nucleic acidsequence and an antisense nucleic acid sequence of the polynucleotideencoding human CXADRL1, GCUD1, or RNF43 protein (SEQ ID NO: 1, 3, or 5).The siRNA is constructed such that a single transcript (double-strandedRNA) has both the sense and complementary antisense sequences from atarget gene, e.g., a hairpin.

The method is used to alter gene expression of a cell, i.e., up-regulatethe expression of CXADRL1, GCUD1, or RNF43, e.g., as a result ofmalignant transformation of the cells. Binding of the siRNA to CXADRL1,GCUD1, or RNF43 transcript in the target cell results in a reduction ofprotein production by the cell. The length of the oligonucleotide is atleast 10 nucleotides and may be as long as the naturally occurringtranscript. Preferably, the oligonucleotide is 19-25 nucleotides inlength. Most preferably, the oligonucleotide is less than 75, 50, or 25nucleotides in length. Examples of CXADRL1, GCUD1, or RNF43 siRNAoligonucleotides which inhibit the expression in mammalian cells includeoligonucleotides containing any of SEQ ID NO: 112-114. These sequencesare target sequence of the following siRNA sequences respectively.

SEQ ID NO: 112, (RNF43);

SEQ ID NO: 113, (RNF43); and

SEQ ID NO: 114, (CXADRL1).

The nucleotide sequence of siRNAs may be designed using an siRNA designcomputer program available from the Ambion website. Nucleotide sequencesfor the siRNA are selected by the computer program based on thefollowing protocol:

Selection of siRNA Target Sites:

-   -   1. Beginning with the AUG start codon of the object transcript,        scan downstream for AA dinucleotide sequences. Record the        occurrence of each AA and the 3′ adjacent 19 nucleotides as        potential siRNA target sites. Tuschl, et al. recommend against        designing siRNA to the 5′ and 3′ untranslated regions (UTRs) and        regions near the start codon (within 75 bases) as these may be        richer in regulatory protein binding sites. UTR-binding proteins        and/or translation initiation complexes may interfere with the        binding of the siRNA endonuclease complex.    -   2. Compare the potential target sites to the human genome        database and eliminate from consideration any target sequences        with significant homology to other coding sequences. The        homology search can be performed using BLAST, which can be found        on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/.    -   3. Select qualifying target sequences for synthesis. At Ambion,        preferably several target sequences can be selected along the        length of the gene for evaluation.

The antisense oligonucleotide or siRNA of the invention inhibit theexpression of the polypeptide of the invention and is thereby useful forsuppressing the biological activity of the polypeptide of the invention.Also, expression-inhibitors, comprising the antisense oligonucleotide orsiRNA of the invention, are useful in the point that they can inhibitthe biological activity of the polypeptide of the invention. Therefore,a composition comprising the antisense oligonucleotide or siRNA of thepresent invention is useful in treating a cell proliferative diseasesuch as cancer.

Moreover, the present invention provides a method for diagnosing a cellproliferative disease using the expression level of the polypeptides ofthe present invention as a diagnostic marker.

This diagnosing method comprises the steps of: (a) detecting theexpression level of the CXADRL1, GCUD1, or RNF43 gene of the presentinvention; and (b) relating an elevation of the expression level to cellproliferative disease, such as cancer.

The expression levels of the the CXADRL1, GCUD1, or RNF43 gene in aparticular specimen can be estimated by quantifying mRNA correspondingto or protein encoded by the CXADRL1, GCUD1, or RNF43 gene.Quantification methods for mRNA are known to those skilled in the art.For example, the levels of mRNAs corresponding to the CXADRL1, GCUD1, orRNF43 gene can be estimated by Northern blotting or RT-PCR. Since thefull-length nucleotide sequences of the CXADRL1, GCUD1, or RNF43 genesare shown in SEQ ID NO: 1, 3, or 5, anyone skilled in the art can designthe nucleotide sequences for probes or primers to quantify the CXADRL1,GCUD1, or RNF43 gene.

Also the expression level of the CXADRL1, GCUD1, or RNF43 gene can beanalyzed based on the activity or quantity of protein encoded by thegene. A method for determining the quantity of the CXADRL1, GCUD1, orRNF43 protein is shown below. For example, immunoassay method is usefulfor the determination of the proteins in biological materials. Anybiological materials can be used for the determination of the protein orit's activity. For example, blood sample is analyzed for estimation ofthe protein encoded by a serum marker. On the other hand, a suitablemethod can be selected for the determination of the activity of aprotein encoded by the CXADRL1, GCUD1, or RNF43 gene according to theactivity of each protein to be analyzed.

Expression levels of the CXADRL1, GCUD1, or RNF43 gene in a specimen(test sample) are estimated and compared with those in a normal sample.When such a comparison shows that the expression level of the targetgene is higher than those in the normal sample, the subject is judged tobe affected with a cell proliferative disease. The expression level ofCXADRL1, GCUD1, or RNF43 gene in the specimens from the normal sampleand subject may be determined at the same time. Alternatively, normalranges of the expression levels can be determined by a statisticalmethod based on the results obtained by analyzing the expression levelof the gene in specimens previously collected from a control group. Aresult obtained by comparing the sample of a subject is compared withthe normal range; when the result does not fall within the normal range,the subject is judged to be affected with the cell proliferativedisease. In the present invention, the cell proliferative disease to bediagnosed is preferably cancer. More preferably, when the expressionlevel of the CXADRL1, or GCUD1 gene is estimated and compared with thosein a normal sample, the cell proliferative disease to be diagnosed isgastric, colorectal, or liver cancer; and when the RNF43 gene isestimated for its expression level, then the disease to be diagnosed iscolorectal, lung, gastric, or liver cancer.

In the present invention, a diagnostic agent for diagnosing cellproliferative disease, such as cancer including gastric, colorectal,lung, and liver cancers, is also provided. The diagnostic agent of thepresent invention comprises a compound that binds to a polynucleotide ora polypeptide of the present invention. Preferably, an oligonucleotidethat hybridizes to the polynucleotide of the present invention, or anantibody that binds to the polypeptide of the present invention may beused as such a compound.

Moreover, the present invention provides a method of screening for acompound for treating a cell proliferative disease using the polypeptideof the present invention. An embodiment of this screening methodcomprises the steps of: (a) contacting a test compound with apolypeptide of the present invention, (b) detecting the binding activitybetween the polypeptide of the present invention and the test compound,and (c) selecting a compound that binds to the polypeptide of thepresent invention.

The polypeptide of the present invention to be used for screening may bea recombinant polypeptide or a protein derived from nature, or a partialpeptide thereof. Any test compound, for example, cell extracts, cellculture supernatant, products of fermenting microorganism, extracts frommarine organism, plant extracts, purified or crude proteins, peptides,non-peptide compounds, synthetic micromolecular compounds, and naturalcompounds, can be used. The polypeptide of the present invention to becontacted with a test compound can be, for example, a purifiedpolypeptide, a soluble protein, a form bound to a carrier, or a fusionprotein fused with other polypeptides.

As a method of screening for proteins, for example, that bind to thepolypeptide of the present invention using the polypeptide of thepresent invention, many methods well known by a person skilled in theart can be used. Such a screening can be conducted by, for example,immunoprecipitation method, specifically, in the following manner. Thegene encoding the polypeptide of the present invention is expressed inanimal cells and so on by inserting the gene to an expression vector forforeign genes, such as pSV2neo, pcDNA I, and pCD8. The promoter to beused for the expression may be any promoter that can be used commonlyand include, for example, the SV40 early promoter (Rigby in Williamson(ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141(1982)), the EF-1α promoter (Kim et al., Gene 91: 217-23 (1990)), theCAG promoter (Niwa et al., Gene 108: 193-9 (1991)), the RSV LTR promoter(Cullen, Methods in Enzymology 152: 684-704 (1987)) the SRα promoter(Takebe et al., Mol Cell Biol 8: 466-72 (1988)), the CMV immediate earlypromoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)),the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94(1982)), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 9:946-58 (1989)), the HSV TK promoter, and so on. The introduction of thegene into animal cells to express a foreign gene can be performedaccording to any methods, for example, the electroporation method (Chuet al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphatemethod (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAEdextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984);Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectinmethod (Derijard, Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)), and soon. The polypeptide of the present invention can be expressed as afusion protein comprising a recognition site (epitope) of a monoclonalantibody by introducing the epitope of the monoclonal antibody, whosespecificity has been revealed, to the N- or C-terminus of thepolypeptide of the present invention. A commercially availableepitope-antibody system can be used (Experimental Medicine 13: 85-90(1995)). Vectors which can express a fusion protein with, for example,β-galactosidase, maltose binding protein, glutathione S-transferase,green florescence protein (GFP), and so on by the use of its multiplecloning sites are commercially available.

A fusion protein prepared by introducing only small epitopes consistingof several to a dozen amino acids so as not to change the property ofthe polypeptide of the present invention by the fusion is also reported.Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, humanc-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag),E-tag (an epitope on monoclonal phage), and such, and monoclonalantibodies recognizing them can be used as the epitope-antibody systemfor screening proteins binding to the polypeptide of the presentinvention (Experimental Medicine 13: 85-90 (1995)).

In immunoprecipitation, an immune complex is formed by adding theseantibodies to cell lysate prepared using an appropriate detergent. Theimmune complex consists of the polypeptide of the present invention, apolypeptide comprising the binding ability with the polypeptide, and anantibody. Immunoprecipitation can be also conducted using antibodiesagainst the polypeptide of the present invention, besides usingantibodies against the above epitopes, which antibodies can be preparedas described above.

An immune complex can be precipitated, for example by Protein Asepharose or Protein G sepharose when the antibody is a mouse IgGantibody. If the polypeptide of the present invention is prepared as afusion protein with an epitope, such as GST, an immune complex can beformed in the same manner as in the use of the antibody against thepolypeptide of the present invention, using a substance specificallybinding to these epitopes, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, forexample, the methods in the literature (Harlow and Lane, Antibodies,511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteinsand the bound protein can be analyzed by the molecular weight of theprotein using gels with an appropriate concentration. Since the proteinbound to the polypeptide of the present invention is difficult to detectby a common staining method, such as Coomassie staining or silverstaining, the detection sensitivity for the protein can be improved byculturing cells in culture medium containing radioactive isotope,³⁵S-methionine or ³⁵S-cystein, labeling proteins in the cells, anddetecting the proteins. The target protein can be purified directly fromthe SDS-polyacrylamide gel and its sequence can be determined, when themolecular weight of a protein has been revealed.

As a method for screening proteins binding to the polypeptide of thepresent invention using the polypeptide, for example, West-Westernblotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used.Specifically, a protein binding to the polypeptide of the presentinvention can be obtained by preparing a cDNA library from cells,tissues, organs (for example, tissues such as testis and ovary forscreening proteins binding to CXADRL1; testis, ovary, and brain forscreening proteins binding to GCUD1; and fetal lung, and fetal kidneyfor those binding to RNF43), or cultured cells expected to express aprotein binding to the polypeptide of the present invention using aphage vector (e.g., ZAP), expressing the protein on LB-agarose, fixingthe protein expressed on a filter, reacting the purified and labeledpolypeptide of the present invention with the above filter, anddetecting the plaques expressing proteins bound to the polypeptide ofthe present invention according to the label. The polypeptide of theinvention may be labeled by utilizing the binding between biotin andavidin, or by utilizing an antibody that specifically binds to thepolypeptide of the present invention, or a peptide or polypeptide (forexample, GST) that is fused to the polypeptide of the present invention.Methods using radioisotope, fluorescence, and such may be also used.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing cells may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-HybridVector System” (Stratagene); the references “Dalton and Treisman, Cell68: 597-612 (1992)”, “Fields and Sternglanz, Trends Genet 10: 286-92(1994)”).

In the two-hybrid system, the polypeptide of the invention is fused tothe SRF-binding region or GAL4-binding region and expressed in yeastcells. A cDNA library is prepared from cells expected to express aprotein binding to the polypeptide of the invention, such that thelibrary, when expressed, is fused to the VP16 or GAL4 transcriptionalactivation region. The cDNA library is then introduced into the aboveyeast cells and the cDNA derived from the library is isolated from thepositive clones detected (when a protein binding to the polypeptide ofthe invention is expressed in yeast cells, the binding of the twoactivates a reporter gene, making positive clones detectable). A proteinencoded by the cDNA can be prepared by introducing the cDNA isolatedabove to E. coli and expressing the protein.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene, and such can be used in addition to the HIS3 gene.

A compound binding to the polypeptide of the present invention can alsobe screened using affinity chromatography. For example, the polypeptideof the invention may be immobilized on a carrier of an affinity column,and a test compound, containing a protein capable of binding to thepolypeptide of the invention, is applied to the column. A test compoundherein may be, for example, cell extracts, cell lysates, etc. Afterloading the test compound, the column is washed, and compounds bound tothe polypeptide of the invention can be prepared.

When the test compound is a protein, the amino acid sequence of theobtained protein is analyzed, an oligo DNA is synthesized based on thesequence, and cDNA libraries are screened using the oligo DNA as a probeto obtain a DNA encoding the protein.

A biosensor using the surface plasmon resonance phenomenon may be usedas a mean for detecting or quantifying the bound compound in the presentinvention. When such a biosensor is used, the interaction between thepolypeptide of the invention and a test compound can be observedreal-time as a surface plasmon resonance signal, using only a minuteamount of polypeptide and without labeling (for example, BIAcore,Pharmacia). Therefore, it is possible to evaluate the binding betweenthe polypeptide of the invention and a test compound using a biosensorsuch as BIAcore.

The methods of screening for molecules that bind when the immobilizedpolypeptide of the present invention is exposed to synthetic chemicalcompounds, or natural substance banks, or a random phage peptide displaylibrary, and the methods of screening using high-throughput based oncombinatorial chemistry techniques (Wrighton et al., Science 273: 458-63(1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9(1996)) to isolate not only proteins but chemical compounds that bind tothe protein of the present invention (including agonist and antagonist)are well known to one skilled in the art.

Alternatively, the screening method of the present invention maycomprise the following steps:

-   -   a) contacting a candidate compound with a cell into which a        vector comprising the transcriptional regulatory region of one        or more marker genes and a reporter gene that is expressed under        the control of the transcriptional regulatory region has been        introduced, wherein the one or more marker genes are selected        from the group consisting of CXADRL1, GCUD1, and RNF43,    -   b) measuring the expression level or activity of said reporter        gene; and    -   c) selecting a compound that reduces the expression level or        activity of said reporter gene as compared to a control.

Suitable reporter genes and host cells are well known in the art. Thereporter construct required for the screening can be prepared using thetranscriptional regulatory region of a marker gene. When thetranscriptional regulatory region of a marker gene is known, a reporterconstruct can be prepared based on the previous sequence information.When the transcriptional regulatory region of a marker gene isunidentified, a nucleotide segment containing the transcriptionalregulatory region can be isolated from a genome library based on thenucleotide sequence information of the marker gene.

A compound isolated by the screening is a candidate for drugs whichpromote or inhibit the activity of the polypeptide of the presentinvention, for treating or preventing diseases attributed to, forexample, cell proliferative diseases, such as cancer. A compound inwhich a part of the structure of the compound obtained by the presentscreening method having the activity of binding to the polypeptide ofthe present invention converted by addition, deletion, insertion, and/orreplacement, is included in the compounds obtained by the screeningmethod of the present invention.

In a further embodiment, the present invention provides methods forscreening candidate agents which are potential targets in the treatmentof cell proliferative disease. As discussed in detail above, bycontrolling the expression levels of the CXADRL1, GCUD1, or RNF43, onecan control the onset and progression of either gastric cancer, orcolorectal, lung, gastric, or liver cancer. Thus, candidate agents,which are potential targets in the treatment of cell proliferativedisease, can be identified through screenings that use the expressionlevels and activities of CXADRL1, GCUD1, or RNF43 as indices. In thecontext of the present invention, such screening may comprise, forexample, the following steps:

-   -   a) contacting a candidate compound with a cell expressing the        CXADRL1, GCUD1, or RNF43; and    -   b) selecting a compound that reduces the expression level of        CXADRL1, GCUD1, or RNF43 in comparison with the expression level        detected in the absence of the test compound.

Cells expressing at least one of the CXADRL1, GCUD1, or RNF43 include,for example, cell lines established from gastric, colorectal, lung, orliver cancers; such cells can be used for the above screening of thepresent invention. The expression level can be estimated by methods wellknown to one skilled in the art. In the method of screening, a compoundthat reduces the expression level of at least one of CXADRL1, GCUD1, orRNF43 can be selected as candidate agents.

In another embodiment of the method for screening a compound fortreating a cell proliferative disease of the present invention, themethod utilizes biological activity of the polypeptide of the presentinvention as an index. Since the CXADRL1, GCUD1, and RNF43 proteins ofthe present invention have the activity of promoting cell proliferation,a compound which promotes or inhibits this activity of one of theseproteins of the present invention can be screened using this activity asan index. This screening method includes the steps of: (a) contacting atest compound with the polypeptide of the present invention; (b)detecting the biological activity of the polypeptide of step (a); and(c) selecting a compound that suppresses the biological activity of thepolypeptide in comparison with the biological activity detected in theabsence of the test compound.

Any polypeptides can be used for screening so long as they comprise thebiological activity of the CXADRL1, GCUD1, or RNF43 protein. Suchbiological activity includes cell-proliferating activity of the humanCXADRL1, GCUD1, or RNF43 protein, and the activity of RNF43 to bind toNOTCH2 or STRIN. For example, a human CXADRL1, GCUD1, or RNF43 proteincan be used and polypeptides functionally equivalent to these proteinscan also be used. Such polypeptides may be endogenously or exogenouslyexpressed by cells.

Any test compounds, for example, cell extracts, cell culturesupernatants, products of fermenting microorganism, extracts of marineorganism, plant extracts, purified or crude proteins, peptides,non-peptide compounds, synthetic micromolecular compounds, and naturalcompounds, can be used.

The compound isolated by this screening is a candidate for agonists orantagonists of the polypeptide of the present invention. The term“agonist” refers to molecules that activate the function of thepolypeptide of the present invention by binding thereto. Likewise, theterm “antagonist” refers to molecules that inhibit the function of thepolypeptide of the present invention by binding thereto. Moreover, acompound isolated by this screening is a candidate for compounds whichinhibit the in vivo interaction of the polypeptide of the presentinvention with molecules (including DNAs and proteins).

When the biological activity to be detected in the present method iscell proliferation, it can be detected, for example, by preparing cellswhich express the polypeptide of the present invention, culturing thecells in the presence of a test compound, and determining the speed ofcell proliferation, measuring the cell cycle and such, as well as bymeasuring the colony forming activity as described in the Examples.

The compound isolated by the above screenings is a candidate for drugswhich inhibit the activity of the polypeptide of the present inventionand can be applied to the treatment of diseases associated with thepolypeptide of the present invention, for example, cell proliferativediseases including cancer. More particularly, when the biologicalactivity of CXADRL1 or GCUD1 protein is used as the index, compoundsscreened by the present method serve as a candidate for drugs for thetreatment of gastric, colorectal, or liver cancer. On the other hand,when the biological activity of RNF43 protein is used as the index,compounds screened by the present method serve as a candidate for drugsfor the treatment of colorectal, lung, gastric, or liver cancer.

Moreover, when the compound isolated by the above screenings is apolypeptide, and a part of the structure of the compound inhibiting theactivity of CXADRL1, GCUD1, or RNF43 protein is converted by addition,deletion, insertion, and/or replacement are also included in thecompounds obtainable by the screening method of the present invention.

In a further embodiment of the method for screening a compound fortreating a cell proliferative disease of the present invention, themethod utilizes the binding ability of RNF43 to NOTCH2 or STRIN. TheRNF43 protein of the present invention was revealed to associated withNOTCH2 and STRIN. These findings suggest that the RNF43 protein of thepresent invention exerts the function of cell proliferation via itsbinding to molecules, such as NOTCH2 and STRIN. Thus, it is expectedthat the inhibition of the binding between the RNF43 protein and NOTCH2or STRIN leads to the suppression of cell proliferation, and compoundsinhibiting the binding serve as pharmaceuticals for treating cellproliferative disease such as cancer. Preferably, the cell proliferativedisease treated by the compound screened by the present method iscolorectal, lung, gastric, or liver cancer.

This screening method includes the steps of: (a) contacting apolypeptide of the present invention with NOTCH2 or STRIN in thepresence of a test compound; (b) detecting the binding between thepolypeptide and NOTCH2 or STRIN; and (c) selecting the compound thatinhibits the binding between the polypeptide and NOTCH2 or STRIN.

The RNF43 polypeptide of the present invention, and NOTCH2 or STRIN tobe used for the screening may be a recombinant polypeptide or a proteinderived from nature, or may also be a partial peptide thereof so long asit retains the binding ability to each other. The RNF43 polypeptide,NOTCH2 or STRIN to be used in the screening can be, for example, apurified polypeptide, a soluble protein, a form bound to a carrier, or afusion protein fused with other polypeptides.

Any test compound, for example, cell extracts, cell culturesupernatants, products of fermenting microorganism, extracts from marineorganism, plant extracts, purified or crude proteins, peptides,non-peptide compounds, synthetic micromolecular compounds, and naturalcompounds, can be used.

As a method of screening for compounds that inhibit the binding betweenthe RNF43 protein and NOTCH2 or STRIN, many methods well known by oneskilled in the art can be used. Such a screening can be carried out asan in vitro assay system, for example, in acellular system. Morespecifically, first, either the RNF43 polypeptide, or NOTCH2 or STRIN isbound to a support, and the other protein is added together with a testsample thereto. Next, the mixture is incubated, washed, and the otherprotein bound to the support is detected and/or measured.

In the same way, a compound interfering the association of CXADRL1 andAIP1 can be isolated by the present invention. It is expected that theinhibition of the binding between the CXADRL1 and AIP1 leads to thesuppression of cell proliferation, and compounds inhibiting the bindingserve as pharmaceuticals for treating cell proliferative disease such ascancer.

Examples of supports that may be used for binding proteins includeinsoluble polysaccharides, such as agarose, cellulose, and dextran; andsynthetic resins, such as polyacrylamide, polystyrene, and silicon;preferably commercial available beads and plates (e.g., multi-wellplates, biosensor chip, etc.) prepared from the above materials may beused. When using beads, they may be filled into a column.

The binding of a protein to a support may be conducted according toroutine methods, such as chemical bonding, and physical adsorption.Alternatively, a protein may be bound to a support via antibodiesspecifically recognizing the protein. Moreover, binding of a protein toa support can be also conducted by means of avidin and biotin binding.

The binding between proteins is carried out in buffer, for example, butare not limited to, phosphate buffer and Tris buffer, as long as thebuffer does not inhibit the binding between the proteins.

In the present invention, a biosensor using the surface plasmonresonance phenomenon may be used as a mean for detecting or quantifyingthe bound protein. When such a biosensor is used, the interactionbetween the proteins can be observed real-time as a surface plasmonresonance signal, using only a minute amount of polypeptide and withoutlabeling (for example, BIAcore, Pharmacia). Therefore, it is possible toevaluate the binding between the RNF43 polypeptide and NOTCH2 or STRINusing a biosensor such as BIAcore.

Alternatively, either the RNF43 polypeptide, or NOTCH2 or STRIN, may belabeled, and the label of the bound protein may be used to detect ormeasure the bound protein. Specifically, after pre-labeling one of theproteins, the labeled protein is contacted with the other protein in thepresence of a test compound, and then, bound proteins are detected ormeasured according to the label after washing.

Labeling substances such as radioisotope (e.g., ³H, ¹⁴C, ³²P, ³³P, ³⁵S,¹²⁵I, ¹³¹I), enzymes (e.g., alkaline phosphatase, horseradishperoxidase, β-galactosidase, β-glucosidase), fluorescent substances(e.g., fluorescein isothiosyanete (FITC), rhodamine), and biotin/avidin,may be used for the labeling of a protein in the present method. Whenthe protein is labeled with radioisotope, the detection or measurementcan be carried out by liquid scintillation. Alternatively, proteinslabeled with enzymes can be detected or measured by adding a substrateof the enzyme to detect the enzymatic change of the substrate, such asgeneration of color, with absorptiometer. Further, in case where afluorescent substance is used as the label, the bound protein may bedetected or measured using fluorophotometer.

Furthermore, the binding of the RNF43 polypeptide and NOTCH2 or STRINcan be also detected or measured using antibodies to the RNF43polypeptide and NOTCH2 or STRIN. For example, after contacting the RNF43polypeptide immobilized on a support with a test compound and NOTCH2 orSTRIN, the mixture is incubated and washed, and detection or measurementcan be conducted using an antibody against NOTCH2 or STRIN.Alternatively, NOTCH2 or STRIN may be immobilized on a support, and anantibody against RNF43 may be used as the antibody.

In case of using an antibody in the present screening, the antibody ispreferably labeled with one of the labeling substances mentioned above,and detected or measured based on the labeling substance. Alternatively,the antibody against the RNF43 polypeptide, NOTCH2, or STRIN, may beused as a primary antibody to be detected with a secondary antibody thatis labeled with a labeling substance. Furthermore, the antibody bound tothe protein in the screening of the present invention may be detected ormeasured using protein G or protein A column.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing cells may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-HybridVector System” (Stratagene); the references “Dalton and Treisman, Cell68: 597-612 (1992)”, “Fields and Sternglanz, Trends Genet 10: 286-92(1994)”).

In the two-hybrid system, the RNF43 polypeptide of the invention isfused to the SRF-binding region or GAL4-binding region and expressed inyeast cells. The NOTCH2 or STRIN binding to the RNF43 polypeptide of theinvention is fused to the VP16 or GAL4 transcriptional activation regionand also expressed in the yeast cells in the existence of a testcompound. When the test compound does not inhibit the binding betweenthe RNF43 polypeptide and NOTCH2 or STRIN, the binding of the twoactivates a reporter gene, making positive clones detectable.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene, and such can be used besides HIS3 gene.

The compound isolated by the screening is a candidate for drugs whichinhibit the activity of the RNF43 protein of the present invention andcan be applied to the treatment of diseases associated with the RNF43protein, for example, cell proliferative diseases such as cancer, moreparticularly colorectal, lung, gastric, or liver cancer. Moreover, whenthe compound isolated by the screening is a polypeptide, and a part ofthe structure of the compound inhibiting the binding between the RNF43protein and NOTCH2 or STRIN is converted by addition, deletion,substitution, and/or insertion are also included in the compoundsobtainable by the screening method of the present invention.

When administrating the compound isolated by the methods of theinvention as a pharmaceutical for humans and other mammals, such asmice, rats, guinea-pigs, rabbits, chicken, cats, dogs, sheep, pigs,cattle, monkeys, baboons, and chimpanzees, for treating a cellproliferative disease (e.g., cancer) the isolated compound can bedirectly administered or can be formulated into a dosage form usingknown pharmaceutical preparation methods. For example, as needed, thedrugs can be taken orally, as sugarcoated tablets, capsules, elixirs andmicrocapsules, or non-orally, in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be mixed withpharmacologically acceptable carriers or medium, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,suspending agents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives, binders, and such, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples of additives that can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum, and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose, and saccharin; flavoringagents such as peppermint, Gaultheria adenothrix oil, and cherry. Whenthe unit dosage form is a capsule, a liquid carrier, such as oil, canalso be further included in the above ingredients. Sterile compositesfor injections can be formulated following normal drug implementationsusing vehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol; polyalcohols, such as propylene glycol andpolyethylene glycol; and non-ionic surfactants, such as Polysorbate 80(TM) and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer and may be formulated with a buffer, such as phosphatebuffer and sodium acetate buffer; a pain-killer, such as procainehydrochloride; a stabilizer, such as benzyl alcohol, phenol; and ananti-oxidant. The prepared injection may be filled into a suitableampule.

Methods well known to one skilled in the art may be used to administerthe inventive pharmaceutical compound to patients, for example asintraarterial, intravenous, percutaneous injections, and also asintranasal, transbronchial, intramuscular, or oral administrations. Thedosage and method of administration vary according to the body-weightand age of a patient and the administration method; however, one skilledin the art can routinely select them. If said compound is encodable by aDNA, the DNA can be inserted into a vector for gene therapy and thevector administered to perform the therapy. The dosage and method ofadministration vary according to the body-weight, age, and symptoms of apatient but one skilled in the art can select them suitably.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with the polypeptide of thepresent invention and regulates its activity is about 0.1 mg to about100 mg per day, preferably about 1.0 mg to about 50 mg per day and morepreferably about 1.0 mg to about 20 mg per day, when administered orallyto a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms, and method of administration, it isconvenient to intravenously inject a dose of about 0.01 mg to about 30mg per day, preferably about 0.1 to about 20 mg per day and morepreferably about 0.1 to about 10 mg per day. Also, in the case of otheranimals too, it is possible to administer an amount converted to 60 kgsof body-weight.

Moreover, the present invention provides a method for treating orpreventing a cell proliferative disease, such as cancer, using anantibody against the polypeptide of the present invention. According tothe method, a pharmaceutically effective amount of an antibody againstthe polypeptide of the present invention is administered. Since theexpression of the CXADRL1, GCUD1, and RNF43 protein are up-regulated incancer cells, and the suppression of the expression of these proteinsleads to the decrease in cell proliferating activity, it is expectedthat cell proliferative diseases can be treated or prevented by bindingthe antibody and these proteins. Thus, an antibody against thepolypeptide of the present invention are administered at a dosagesufficient to reduce the activity of the protein of the presentinvention, which is in the range of 0.1 to about 250 mg/kg per day. Thedose range for adult humans is generally from about 5 mg to about 17.5g/day, preferably about 5 mg to about 10 g/day, and most preferablyabout 100 mg to about 3 g/day.

Alternatively, an antibody binding to a cell surface marker specific fortumor cells can be used as a tool for drug delivery. For example, theantibody conjugated with a cytotoxic agent is administered at a dosagesufficient to injure tumor cells.

The present invention also relates to a method of inducing anti-tumorimmunity comprising the step of administering CXADRL1, GCUD1, or RNF43protein, an immunologically active fragment thereof, or a polynucleotideencoding the protein or fragments thereof. The CXADRL1, GCUD1, or RNF43protein, or the immunologically active fragments thereof are useful asvaccines against cell proliferative diseases. In some cases the proteinsor fragments thereof may be administered in a form bound to the T cellreceptor (TCR) or presented by an antigen presenting cell (APC), such asmacrophage, dendritic cell (DC), or B-cells. Due to the strong antigenpresenting ability of DC, the use of DC is most preferable among theAPCs.

In the present invention, vaccine against cell proliferative diseaserefers to a substance that has the function to induce anti-tumorimmunity upon inoculation into animals. According to the presentinvention, polypeptides comprising the amino acid sequence of SEQ ID NO:80, 97, or 108 were suggested to be HLA-A24 or HLA-A*0201 restrictedepitope peptides that may induce potent and specific immune responseagainst colorectal, lung, gastric, or liver cancer cells expressingRNF43. According to the present invention, polypeptides comprising theamino acid sequence of SEQ ID NO: 124 was suggested to be HLA-A*0201restricted epitopes peptides that may induce potent and specific immuneresponse against colorectal, gastric, or liver cancer cells expressingCXADRL1. According to the present invention, polypeptides comprising theamino acid sequence of SEQ ID NO: 164 was suggested to be HLA-A*0201restricted epitopes peptides that may induce potent and specific immuneresponse against colorectal, gastric, or liver cancer cells expressingGCUD1. Thus, the present invention also encompasses method of inducinganti-tumor immunity using polypeptides comprising the amino acidsequence of SEQ ID NO: 80, 97, 108, 124 or 164.

Furthermore, the present invention revealed that modification of anchorresidues increases the binding affinity of epitope peptides to HLAs.Therefore, polypeptides administered for treating or preventing cancer,or inducing anti-tumor immunity according to the present inventionincludes epitope peptides wherein the anchor residues are modified.Herein, the phrase “anchor residue(s)” refers to amino acid residues ofthe epitope that binds to the HLA class I peptide-binding cleft, butthat does not contact with TCR; more specifically, positions two andnine of an epitope peptide is considered to be such anchor residues.According to the HLA-A2 antigen motif previously reported by Smith etal. (Smith et al., Mol Immunol 35: 1033-43 (1998)), leucine (Leu) andisoleucine (Iso) have proven to be an optimal anchor residue at position2 that enhance the binding affinity of the peptide for the HLA-A*0201molecule. Similarly, valine (Val) at position 9 is also preferred fornonamer peptides. Thus, nonamer peptides wherein the amino acid atposition two is Leu or Ile, or the amino acid at position nine is Leu,Ile, or Val are preferred examples as the polypeptide to be administeredaccording to the present invention. Particularly preferable examples ofsuch nonamer peptides include those having the amino acid sequence ofSEQ ID NOs: 195-198. However, the present invention is not restricted tothese examples and any modification may be introduced into thepolypeptides used for the present invention.

In general, anti-tumor immunity includes immune responses such asfollows:

-   induction of cytotoxic lymphocytes against tumors,-   induction of antibodies that recognize tumors, and-   induction of anti-tumor cytokine production.

Therefore, when a certain protein induces any one of these immuneresponses upon inoculation into an animal, the protein is decided tohave anti-tumor immunity inducing effect. The induction of theanti-tumor immunity by a protein can be detected by observing in vivo orin vitro the response of the immune system in the host against theprotein.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. A foreign substance that enters the livingbody is presented to T cells and B cells by the action of antigenpresenting cells (APCs). T cells that respond to the antigen presentedby APC in antigen specific manner differentiate into cytotoxic T cells(or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen,and then proliferate (this is referred to as activation of T cells).Therefore, CTL induction by a certain peptide can be evaluated bypresenting the peptide to T cell by APC, and detecting the induction ofCTL. Furthermore, APC has the effect of activating CD4+ T cells, CD8+ Tcells, macrophages, eosinophils, and NK cells. Since CD4+ T cells andCD8+ T cells are also important in anti-tumor immunity, the anti-tumorimmunity inducing action of the peptide can be evaluated using theactivation effect of these cells as indicators.

A method for evaluating the inducing action of CTL using dendritic cells(DCs) as APC is well known in the art. DC is a representative APC havingthe strongest CTL inducing action among APCs. In this method, the testpolypeptide is initially contacted with DC, and then this DC iscontacted with T cells. Detection of T cells having cytotoxic effectsagainst the cells of interest after the contact with DC shows that thetest polypeptide has an activity of inducing cytotoxic T cells. Activityof CTL against tumors can be detected, for example, using the lysis of⁵¹Cr-labeled tumor cells as the indicator. Alternatively, the method ofevaluating the degree of tumor cell damage using ³H-thymidine uptakeactivity or LDH (lactose dehydrogenase)-release as the indicator is alsowell known.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also beused as the APC. The induction of CTL is reported that it can beenhanced by culturing PBMC in the presence of GM-CSF and IL-4.Similarly, CTL has been shown to be induced by culturing PBMC in thepresence of keyhole limpet hemocyanin (KLH) and IL-7.

The test polypeptides confirmed to possess CTL inducing activity bythese methods are polypeptides having DC activation effect andsubsequent CTL inducing activity. Therefore, polypeptides that induceCTL against tumor cells are useful as vaccines against tumors.Furthermore, APC that acquired the ability to induce CTL against tumorsby contacting with the polypeptides are useful as vaccines againsttumors. Furthermore, CTL that acquired cytotoxicity due to presentationof the polypeptide antigens by APC can be also used as vaccines againsttumors. Such therapeutic methods for tumors using anti-tumor immunitydue to APC and CTL are referred to as cellular immunotherapy.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction is known to increase by combining aplurality of polypeptides having different structures and contactingthem with DC. Therefore, when stimulating DC with protein fragments, itis advantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of anti-tumor immunity by a polypeptide canbe confirmed by observing the induction of antibody production againsttumors. For example, when antibodies against a polypeptide are inducedin a laboratory animal immunized with the polypeptide, and when growthof tumor cells is suppressed by those antibodies, the polypeptide can bedetermined to have an ability to induce anti-tumor immunity.

Anti-tumor immunity is induced by administering the vaccine of thisinvention, and the induction of anti-tumor immunity enables treatmentand prevention of cell proliferating diseases, such as gastric,colorectal, lung, and liver cancers. Therapy against cancer orprevention of the onset of cancer includes any of the steps, such asinhibition of the growth of cancerous cells, involution of cancer, andsuppression of occurrence of cancer. Decrease in mortality ofindividuals having cancer, decrease of tumor markers in the blood,alleviation of detectable symptoms accompanying cancer, and such arealso included in the therapy or prevention of cancer. Such therapeuticand preventive effects are preferably statistically significant. Forexample, in observation, at a significance level of 5% or less, whereinthe therapeutic or preventive effect of a vaccine against cellproliferative diseases is compared to a control without vaccineadministration. For example, Student's t-test, the Mann-Whitney U-test,or ANOVA may be used for statistical analyses.

The above-mentioned protein having immunological activity or a vectorencoding the protein may be combined with an adjuvant. An adjuvantrefers to a compound that enhances the immune response against theprotein when administered together (or successively) with the proteinhaving immunological activity. Examples of adjuvants include choleratoxin, salmonella toxin, alum, and such, but are not limited thereto.Furthermore, the vaccine of this invention may be combined appropriatelywith a pharmaceutically acceptable carrier. Examples of such carriersare sterilized water, physiological saline, phosphate buffer, culturefluid, and such. Furthermore, the vaccine may contain as necessary,stabilizers, suspensions, preservatives, surfactants, and such. Thevaccine is administered systemically or locally. Vaccine administrationmay be performed by single administration, or boosted by multipleadministrations.

When using APC or CTL as the vaccine of this invention, tumors can betreated or prevented, for example, by the ex vivo method. Morespecifically, PBMCs of the subject receiving treatment or prevention arecollected, the cells are contacted with the polypeptide ex vivo, andfollowing the induction of APC or CTL, the cells may be administered tothe subject. APC can be also induced by introducing a vector encodingthe polypeptide into PBMCs ex vivo. APC or CTL induced in vitro can becloned prior to administration. By cloning and growing cells having highactivity of damaging target cells, cellular immunotherapy can beperformed more effectively. Furthermore, APC and CTL isolated in thismanner may be used for cellular immunotherapy not only againstindividuals from whom the cells are derived, but also against similartypes of tumors from other individuals.

Furthermore, a pharmaceutical composition for treating or preventing acell proliferative disease, such as cancer, comprising apharmaceutically effective amount of the polypeptide of the presentinvention is provided. The pharmaceutical composition may be used forraising anti-tumor immunity. The normal expression of CXADRL1,restricted to testis and ovary; normal expression of GCUD1 is restrictedto testis, ovary, and brain; and normal expression of RNF43 isrestricted to fetus, more specifically to fetal lung and kidney.Therefore, suppression of these genes may not adversely affect otherorgans. Thus, the CXADRL1 and GCUD1 polypeptides are preferable fortreating cell proliferative disease, especially gastric, colorectal, orliver cancer; and RNF43 polypeptide is preferable for treating cellproliferative disease, especially colorectal, lung, gastric, and livercancers.

Furthermore, since peptide fragments of RNF43 comprising the amino acidsequences of SEQ ID NO: 80, 97, and 108, respectively, were revealed toinduce immune response against RNF43, polypeptides comprising the aminoacid sequence of SEQ ID NO: 80, 97, or 108 are preferable examples ofpolypeptides that can be used in a pharmaceutical composition fortreating or preventing cell proliferative disease, especiallycolorectal, lung, gastric, and liver cancers.

Furthermore, since peptide fragments of CXADRL1 comprising the aminoacid sequences of SEQ ID NO: 124, was revealed to induce immune responseagainst CXADRL1, polypeptide comprising the amino acid sequence of SEQID NO: 124 is a preferable example of polypeptide that can be used in apharmaceutical composition for treating or preventing cell proliferativedisease, especially colorectal, lung, gastric, and liver cancers.Moreover, anchor-modified polypeptides of the polypeptide comprising theamino acid sequence of SEQ ID NO: 124 were revealed to exhibit increasedbinding affinity to HLA-A*0201 molecules, activate a certain portion ofTCR repertoire recognizing the naturally processed wild-type epitopepeptide presented by tumor cells, and elicit native peptide specificCTSs more frequently and abundantly than the wild-type peptide. Thus,such anchor-modified polypeptides are preferable examples of polypeptidethat can be used in a pharmaceutical composition for treating orpreventing the cell proliferative diseases. Example of theseanchor-modified polypeptides includes those having the amino acidsequences of SEQ ID NOs: 195 and 196.

Furthermore, since peptide fragment of GCUD1 comprising the amino acidsequences of SEQ ID NO: 164 was revealed to induce immune responseagainst GCUD1, polypeptide comprising the amino acid sequence of SEQ IDNO: 164 is a preferable example of polypeptide that can be used in apharmaceutical composition for treating or preventing cell proliferativedisease, especially colorectal, lung, gastric, and liver cancers.Moreover, anchor-modified polypeptide of the polypeptides comprising theamino acid sequence of SEQ ID NO: 164 were revealed to exhibit increasedbinding affinity to HLA-A*0201 molecules, activate a certain portion ofTCR repertoire recognizing the naturally processed wild-type epitopepeptide presented by tumor cells, and elicit native peptide specificCTSs more frequently and abundantly than the wild-type peptide. Thus,such anchor-modified polypeptides are preferable examples of polypeptidethat can be used in a pharmaceutical composition for treating orpreventing the cell proliferative diseases. Example of theseanchor-modified polypeptides includes those having the amino acidsequences of SEQ ID NOs: 197 and 198.

In the present invention, the polypeptide or fragment thereof isadministered at a dosage sufficient to induce anti-tumor immunity, whichis in the range of 0.1 mg to 10 mg, preferably 0.3 mg to 5 mg, morepreferably 0.8 mg to 1.5 mg. The administrations are repeated. Forexample, 1 mg of the peptide or fragment thereof may be administered 4times in every two weeks for inducing the anti-tumor immunity.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Any patents, patent applications, andpublications cited herein are incorporated by reference.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is illustrated in details by following Examples,but is not restricted to these Examples.

1. Materials and Methods (1) Patients and Tissue Specimens

All gastric and colorectal cancer tissues, as well as correspondingnon-cancerous tissues were obtained with informed consent from surgicalspecimens of patients who underwent surgery.

(2) Genome-Wide cDNA Microarray

In-house genome-wide cDNA microarray comprising 23040 genes were used inthis study. DNase I treated total RNA extracted from microdissectedtissue was amplified with Ampliscribe T7 Transcription Kit (EpicentreTechnologies) and labeled during reverse transcription with Cy-dye(Amersham) (RNA from non-cancerous tissue with Cy5 and RNA from tumorwith Cy3). Hybridization, washing, and detection were carried out asdescribed previously (Ono et al., Cancer Res. 60: 5007-11 (2000)), andfluorescence intensity of Cy5 and Cy3 for each target spot was measuredusing Array Vision software (Amersham Pharmacia). After subtraction ofbackground signal, duplicate values were averaged for each spot. Then,all fluorescence intensities on a slide were normalized to adjust themean Cy5 and Cy3 intensity of 52 housekeeping genes for each slide.Genes with intensities below 25,000 fluorescence units for both Cy3 andCy5 were excluded from further investigation, and those with Cy3/Cy5signal ratios>2.0 were selected for further evaluation.

(3) Cell Lines

Human embryonic kidney 293 (HEK293) were obtained from TaKaRa. COS7cell, NIH3T3 cell, human cervical cancer cell line HeLa, human gastriccancer cell lines MKN-1 and MKN-28, human hepatoma cell line Alexander,and human colon cancer cell lines, LoVo, HCT 116, DLD-1 and SW480, wereobtained from the American Type Culture Collection (ATCC, Rockville,Md.). Human hepatoma cell line SNU475 and human colon cancer cell lines,SNUC4 and SNUC5, were obtained from the Korea cell-line bank. All cellswere grown in monolayers in appropriate media: Dulbecco's modifiedEagle's medium for COS7, NIH3T3, HEK293, and Alexander; RPMI1640 forMKN-1, MKN-28, SNU475, SNUC4, DLD-1 and SNUC5; McCoy's 5A medium forHCT116; Leibovitz's L-15 for SW480; HAM's F-12 for LoVo; and Eagle'sminimum essential medium for HeLa (Life Technologies, Grand Island,N.Y.). All media were supplemented with 10% fetal bovine serum and 1%antibiotic/antimycotic solution (Sigma). A human gastric cancer cellline St-4 was kindly provided by Dr. Tsuruo of Cancer Institute inJapan. St-4 cells were grown in monolayers in RPMI1640 supplemented with10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma).T2 cells (HLA-A*0201) and EHM (HLA-A3/3), human B-lymphoblastoid celllines, were generous gifts from Prof Shiku (Univ. Mie). HT29 (coloncarcinoma cell line; HLA-A24/01), WiDR (colon carcinoma cell line;HLA-A24/01), HCT116 (colon carcinoma cell line; HLA-A02/01), DLD-1(colon carcinoma cell line; HLA-A24/01), SNU475 (hepatocellularcarcinoma cell line; HLA-A*0201), MKN45 (gastric cancer cell line;HLA-A2 negative), and MKN74 (gastric cancer cell line; HLA-A2 negative)were also purchased from ATCC. TISI cells (HLA-A24/24) were generousgifts from Takara Shuzo Co, Ltd. (Otsu, Japan). RT-PCR examinationsrevealed strong CXADRL1 expression in SNU475 and MKN74, and strong GCUD1expression in SNU475 and MKN45.

(4) RNA Preparation and RT-PCR

Total RNA was extracted with Qiagen RNeasy kit (Qiagen) or Trizolreagent (Life Technologies) according to the manufacturers' protocols.Ten-microgram aliquots of total RNA were reversely transcribed forsingle-stranded cDNAs using poly dT₁₂₋₁₈ primer (Amersham PharmaciaBiotech) with Superscript II reverse transcriptase (Life Technologies).Each single-stranded cDNA preparation was diluted for subsequent PCRamplification by standard RT-PCR experiments carried out in 20 μlvolumes of PCR buffer (TaKaRa). Amplification was conducted underfollowing conditions: denaturing for 4 min at 94° C., followed by 20(for GAPDH), 35 (for CXADRL1), 30 (for GCUD1), 30 (for RNF43) cycles of94° C. for 30 s, 56° C. for 30 s, and 72° C. for 45 s, in GeneAmp PCRsystem 9700 (Perkin-Elmer, Foster City, Calif.). Primer sequences were;for GAPDH: forward, 5′-ACAACAGCCTCAAGATCATCAG (SEQ ID NO: 7) andreverse, 5′-GGTCCACCACTGACACGTTG (SEQ ID NO: 8); for CXADRL1: forward,5′-AGCTGAGACATTTGTTCTCTTG (SEQ ID NO: 9) and reverse: 5′-TATAAACCAGCTGAGTCCAGAG (SEQ ID NO: 10); for GCUD1 forward:5′-TTCCCGATATCAACATCTACCAG (SEQ ID NO: 11) reverse:5′-AGTGTGTGACCTCAATAAGGCAT (SEQ ID NO: 12), for RNF43 forward;5′-CAGGCTTTGGACGCACAGGACTGGTAC-3′ (SEQ ID NO: 13) and reverse;5′-CTTTGTGATCATCCTGGCTTCGGTGCT-3′ (SEQ ID NO: 14).

(5) Northern-Blot Analysis

Human multiple-tissue blots (Clontech, Palo Alto, Calif.) werehybridized with ³²P-labeled PCR products of CXADRL1, GCUD1, or RNF43.Pre-hybridization, hybridization and washing were performed according tothe supplier's recommendations. The blots were autoradiographed withintensifying screens at −80° C. for 24 to 72 h.

(6) 5′ Rapid Amplification of cDNA Ends (5′ RACE)

5′ RACE experiments were carried out using Marathon cDNA amplificationkit (Clontech) according to the manufacturer's instructions. For theamplification of the 5′ part of CXADRL1, gene-specific reverse primers(5′-GGTTGAGATTTAAGTTCTCAAA-3′ (SEQ ID NO: 15)) and the AP-1 primersupplied with the kit were used. The cDNA template was synthesized fromhuman testis mRNA (Clontech). The PCR products were cloned using TAcloning kit (Invitrogen) and their sequences were determined with ABIPRISM 3700 DNA sequencer (Applied Biosystems).

(7) Construction of Plasmids Expressing CXADRL1, GCUD1, and FLJ20315

The entire coding regions of CXADRL1, GCUD1, and RNF43 were amplified byRT-PCR using gene specific primer sets; for CXADRL1,5′-AGTTAAGCTTGCCGGGATGACTTCTCAGCGTTCCCCTCTGG-3′ (SEQ ID NO: 16) and5′-ATCTCGAGTACCAAGGACCCGGCCCGACTCTG-3′ (SEQ ID NO: 17); for GCUD15′-GCGGATCCAGGATGGCTGCTGCAGCTCCTCCAAG-3′ (SEQ ID NO: 18) and5′-TAGAATTCTTAAAGAACTTAATCTCCGTGTCAACAC-3′ (SEQ ID NO: 19); and forRNF43, 5′-TGCAGATCTGCAGCTGGTAGCATGAGTGGTG-3′ (SEQ ID NO: 20) and5′-GAGGAGCTGTGTGAACAGGCTGTGTGAGATGT-3′ (SEQ ID NO: 21). The PCR productswere cloned into appropriate cloning site of either pcDNA3.1(Invitrogen), or pcDNA3.1myc/His (Invitrogen) vector.

(8) Immunoblotting

Cells transfected with pcDNA3.1myc/His-CXADRL1, pcDNA3.1myc/His-GCUD1,pcDNA3.1myc/His-RNF43, or pcDNA3.1myc/His-LacZ were washed twice withPBS and harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mMTris-HCl pH 7.4, 1 mM DTT, and 1× complete Protease Inhibitor Cocktail(Boehringer)). Following homogenization, the cells were centrifuged at10,000×g for 30 min, the supernatant were standardized for proteinconcentration by the Bradford assay (Bio-Rad). Proteins were separatedby 10% SDS-PAGE and immunoblotted with mouse anti-myc (SANTA CRUZ)antibody. HRP-conjugated goat anti-mouse IgG (Amersham) served as thesecondary antibody for the ECL Detection System (Amersham).

(9) Immunohistochemical Staining

Cells transfected with pcDNA3.1myc/His-CXADRL1, pcDNA3.1myc/His-GCUD1,pcDNA3.1myc/His-RNF43, or pcDNA3.1myc/His-LacZ were fixed with PBScontaining 4% paraformaldehyde for 15 min, then made permeable with PBScontaining 0.1% Triton X-100 for 2.5 min at RT. Subsequently the cellswere covered with 2% BSA in PBS for 24 h at 4° C. to block non-specifichybridization. Mouse anti-myc monoclonal antibody (Sigma) at 1:1000dilution was used as the primary antibody, and the reaction wasvisualized after incubation with Rhodamine-conjugated anti-mousesecondary antibody (Leinco and ICN). Nuclei were counter-stained with4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescentimages were obtained under an ECLIPSE E800 microscope.

(10) Colony Formation Assay

Cells transfected with plasmids expressing each gene or control plasmidswere incubated with an appropriate concentration of geneticin for 10 to21 days. The cells were fixed with 100% methanol and stained by Giemsasolution. All experiments were carried out in duplicate.

(II) Establishment of Cells Over-Expressing CXADRL1 or RNF43

NIH3T3, COS7, and LoVo cells transfected with eitherpcDNA3.1myc/His-CXADRL1, pcDNA3.1myc/His-RNF43, pcDNA3.1myc/His-LacZ, orcontrol plasmids, respectively, were maintained in media containingappropriate concentration of geneticin. Two weeks after thetransfection, surviving single colonies were selected, and expression ofeach gene was examined by semi-quantitative RT-PCR.

(12) Examination on the Effect of Anti-Sense Oligonucleotides on CellGrowth

Cells plated onto 10-cm dishes (2×10⁵ cells/dish) were transfectedeither with plasmid, or synthetic S-oligonucleotides of CXADRL1, GCUD1,or RNF43 using LIPOFECTIN Reagent (GIBCO BRL). Then the cells werecultured with the addition of an appropriate concentration of geneticinfor six to twelve days. The cells were then fixed with 100% methanol andstained by Giemsa solution. Sequences of the S-oligonucleotides were asfollows:

(SEQ ID NO: 22) CXADRL1-S4, 5′-TCTGCACGGTGAGTAG-3′; (SEQ ID NO: 23)CXADRL1-AS4, 5′-CTACTCACCGTGCAGA-3′; (SEQ ID NO: 24) CXADRL1-S5,5′-TTCTGTAGGTGTTGCA-3′; (SEQ ID NO: 25) CXADRL1-AS5,5′-TGCAACACCTACAGAA-3′; (SEQ ID NO: 26) GCUD1-S5,5′-CTTTTCAGGATGGCTG-3′; (SEQ ID NO: 27) GCUD1-AS5,5′-CAGCCATCCTGAAAAG-3′; (SEQ ID NO: 28) GCUD1-S8,5′-AGGTTGAGGTAAGCCG-3′; (SEQ ID NO: 29) GCUD1-AS8,5′-CGGCTTACCTCAACCT-3′; (SEQ ID NO: 30) RNF43-S1,5′-TGGTAGCATGAGTGGT-3′; and (SEQ ID NO: 31) RNF43-AS1,5′-ACCACTCATGCTACCA-3′.

(13) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)Assay

Cells plated at a density of 5×10⁵ cells/100 mm dish were transfected intriplicate with sense or antisense S-oligonucleotides designated tosuppress the expression of CXADRL1, GCUD1, or RNF43. Seventy-two hoursafter transfection, the medium was replaced with fresh medium containing500 μg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) (Sigma) and the plates were incubated for four hours at 37° C.Subsequently, the cells were lysed by the addition of 1 ml of 0.01 NHCl/10% SDS and the absorbance of lysates was measured with ELISA platereader at a test wavelength of 570 nm (reference, 630 nm). The cellviability was represented by the absorbance compared to that of controlcells.

(14) Construction of psiH1BX3.0

Since H1RNA gene was reported to be transcribed by RNA polymerase III,which produce short transcripts with uridines at the 3′ end, a genomicfragment of H1RNA gene containing its promoter region was amplified byPCR using a set of primers [5′-TGGTAGCCAAGTGCAGGTTATA-3′ (SEQ ID NO:32), and 5′-CCAAAGGGTTTCTGCAGTTTCA-3′ (SEQ ID NO: 33)] and humanplacental DNA as a template. The products were purified and cloned intopCR2.0 plasmid vector using TA cloning kit (Invitrogen) according to thesupplier's protocol. The BamHI and XhoI fragment containing the H1RNAgene was purified and cloned into pcDNA3.1(+) plasmid at the nucleotideposition from 1257 to 56, which plasmid was amplified by PCR with a setof primers, 5′-TGCGGATCCAGAGCAGATTGTACTGAGAGT-3′ (SEQ ID NO: 34) and5′-CTCTATCTCGAGTGAGGCGGAAAGAACCA-3′ (SEQ ID NO: 35), and then digestedwith BamHI and XhoI. The ligated DNA was used as a template for PCR withprimers, 5′-TTTAAGCTTGAAGACCATTTTTGGAAAAAAAAAAAAAAAAAAAAAAC-3′ (SEQ IDNO: 36) and 5′-TTTAAGCTTGAAGACATGGGAAAGAGTGGTCTCA-3′ (SEQ ID NO: 37).The product was digested with HindIII, and subsequently self-ligated toproduce psiH1BX3.0 vector plasmid. As the control, psiH1BX-EGFP wasprepared by cloning double-stranded oligonucleotides of5′-CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-3′ (SEQ ID NO:38) and 5′-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-3′ (SEQID NO: 39) into the BbsI site of the psiH1BX vector.

(15) Examination on the Gene Silencing Effect of RNF43-, orCXADRL1-siRNAs

A plasmid expressing either RNF43-siRNA or CXADRL1-siRNA was prepared bycloning double-stranded oligonucleotides into psiH1BX3.0 vector.Oligonucleotides used as RNF43 siRNAs were:5′-TCCCGTCACCGGATCCAACTCAGTTCAAGAGACTGAGTTGGATCCGGTGAC-3′ (SEQ ID NO:40) and 5′-AAAAGTCACCGGATCCAACTCAGTCTCTTGAACTGAGTTGGATCCGGTGAC-3′ (SEQID NO: 41) as siRNA16-4;5′-TCCCGCTATTGCACAGAACGCAGTTCAAGAGACTGCGTTCTGTGCAATAGC-3′ (SEQ ID NO:42) and 5′-AAAAGCTATTGCACAGAACGCAGTCTCTTGAACTGCGTTCTGTGCAATAGC-3′ (SEQID NO: 43) as siRNA1834;5′-TCCCCAGAAAGCTGTTATCAGAGTTCAAGAGACTCTGATAACAGCTTTCTG-3′ (SEQ ID NO:44) and 5′-AAAACAGAAAGCTGTTATCAGAGTCTCTTGAACTCTGATAACAGCTTTCTG-3′ (SEQID NO: 45) as siRNA1;5′-TCCCTGAGCCACCTCCAATCCACTTCAAGAGAGTGGATTGGAGGTGGCTCA-3′ (SEQ ID NO:46) and 5′-AAAATGAGCCACCTCCAATCCACTCTCTTGAAGTGGATTGGAGGTGGCTCA-3′ (SEQID NO: 47) as siRNA14;5′-TCCCCTGCACGGACATCAGCCTATTCAAGAGATAGGCTGATGTCCGTGCAG-3′ (SEQ ID NO:48) and 5′-AAAACTGCACGGACATCAGCCTATCTCTTGAATAGGCTGATGTCCGTGCAG-3′ (SEQID NO: 49) as siRNA15. Oligonucleotides used as CXADRL1-siRNAs were:5′-TCCCGTGTCAGAGAGCCCTGGGATTCAAGAGATCCCAGGGCTCTCTGACAC-3′ (SEQ ID NO:50) and 5′-AAAAGTGTCAGAGAGCCCTGGGATCTCTTGAATCCCAGGGC TCTCTGACAC-3′ (SEQID NO: 51) as siRNA#1; 5′-TCCCCCTCAATGTCATTTGGATGTTCAAGAGACATCCAAATGCAATTGAGG-3′ (SEQ ID NO: 52) and 5′-AAAACCTCAATGTCATTTGGATGTCTCTTGAACATCCAAATGCAATTGAGG-3′ (SEQ ID NO: 5 3) assiRNA#2; 5′-TCCCTGTCATTTGGATGGTCACTTTCAAGAGAAGTGACCATCCA AATGACA-3′ (SEQID NO: 54) and 5′-AAAATGTCATTTGGATGGTCACTTCTCTTGAAAGTGACCATCCAAATGACA-3′ (SEQ ID NO: 55) as siRNA#3; 5′-TCCCTGCCAACCAACCTGAACAGTTCAAGAGACTGTTCAGGTTGGTTGGCA-3′ (SEQ ID NO: 56) and5′-AAAATGCCAACCAACCTGAACAGTCTCTTGAACTGTTCAGGTTGGTTGGCA-3′ (SEQ ID NO:57) as siRNA#4; 5′-TCCCCCAACCTGAACAGGTCATCTTCAAGAGAGATGACCTGTTCAGGTTGG-3′ (SEQ ID NO: 58) and 5′-AAAACCAACCTGAACAGGTCATCTCTCTTGAAGATGACCTGTTCAGGTTGG-3′ (SEQ ID NO: 59) as siRNA#5;5′-TCCCCCTGAACAGGTCATCCTGTTTCAAGAGAACAGGATGACCTGTTCAGG-3′ (SEQ ID NO:60) and 5′-AAAACCTGAACAGGTCATCCTGTTCTCTTGAAACAGGA TGACCTGTTCAGG-3′ (SEQID NO: 61) as siRNA#6; and 5′-TCCCCAGGTCATCCTGTATCAGGTTCAAGAGACCTGATACAGGATGACCTG-3′ (SEQ ID NO: 62) and 5′-AAAACAGGTCATCCTGTATCAGGTCTCTTGAACCTGATACAGGATGACCTG-3′ (SEQ ID NO: 63) asCXADRL-siRNA#7. psiH1BX-RNF43, psiH1BX-CXADRL1, or psiH1 BX-mockplasmids were transfected into SNUC4 or St-4 cells using FuGENE6 reagent(Roche) according to the supplier's recommendations. Total RNA wasextracted from the cells 48 hours after the transfection.

(16) Construction of Recombinant Amino- and Carboxyl-Terminal Regions ofRNF43 Protein

The amino- and carboxyl-terminal regions of RNF43 was amplified byRT-PCR using following sets of primers:5′-GAAGATCTGCAGCGGTGGAGTCTGAAAG-3′ (SEQ ID NO: 64) and5′-GGAATTCGGACTGGGAAAATGAATCTCCCTC-3′ (SEQ ID NO: 65) for theamino-terminal region; and 5′-GGAGATCTCCTGATCAGCAAGTCACC-3′ (SEQ ID NO:66) and 5′-GGAATTCCACAGCCTGTTCACACAGCTCCTC-3′ (SEQ ID NO: 67) for thecarboxyl-terminal region. The products were digested with BamHI-EcoRIand cloned into the BamHI-EcoRI site of pET-43.1a(+) vector (Novagen).The plasmids were transfected into E. coli BL21trxB(DE3)pLysS cells(Stratagene). Recombinant RNF43 protein was extracted from cellscultured at 25° C. for 16 h after the addition of 0.2 mM IPTG.

(17) Yeast Two-Hybrid Experiment

Yeast two-hybrid assay was performed using MATCHMAKER GAL4 Two-HybridSystem 3 (Clontech) according to the manufacturer's protocols. Theentire coding sequence of RNF43 was cloned into the EcoR I-BamH I siteof pAS2-1 vector as a bait for screening human-testis cDNA library(Clontech). To confirm the interaction in yeast, pAS2-RNF43 was used asbait vector, pACT2-NOTCH2 and pACT2-STRIN as prey vector.

Furthermore, the cytoplasmic region of CXADRL1 was cloned into the EcoRIsite of pAS2-1 vector as a bait for screening human testis cDNA library(Clontech). To confirm interaction in yeast, pAS2-CXADRL1 was used asthe bait vector, and pACT2-AIP1 as the prey vector.

(18) Preparation of CXADRL Specific Antibody

Anti-CXADRL antisera were prepared by immunization with syntheticpolypeptides of CXADRL1 encompassing codons from 235 to 276 for Ab-1,from 493 to 537 for Ab-2, or from 70 to 111 for Ab-3. Sera were purifiedusing recombinant His-tagged CXADRL1 protein prepared in E. colitransfected with pET-CXADRL plasmid. Protein extracted from cellsexpressing Flag-tagged CXADRL1 was further separated by 10% SDS-PAGE andimmunoblotted with either anti-CXADRL1 sera or anti-Flag antibody.HRP-conjugated goat anti-rabbit IgG or HRP-conjugated sheep anti-mouseIgG antibody served as the secondary antibody, respectively, for ECLDetection System (Amersham Pharmacia Biotech, Piscataway, N.J.).Immunoblotting with anti-CXADRL antisera showed a 50 kD band ofFLAG-tagged CXADRL1, which pattern was identical to that detected withanti-FLAG antibody.

(19) Preparation of Recombinant GCUD1 Protein

To generate an antibody specific against GCUD1, recombinant GCUD1protein was prepared. The entire coding region of GCUD1 was amplified byRT-PCR with a set of primers, 5′-GCGGATCCAGGATGGCTGCAGCTCCTCCAAG-3′ (SEQID NO: 68) and 5′-CTGAATTCACTTAAAGAACTTAATCTCCGTGTCAACAC-3′ (SEQ ID NO:69). The product was purified, digested with BamH1 and EcoR1, and clonedinto an appropriate cloning site of pGEX6P-2. The resulting plasmid wasdubbed pGEX-GCUD1. pGEX-GCUD1 plasmid was transformed into E. coliDH10B. The production of the recombinant protein was induced by theaddition of IPTG, and the protein was purified with GlutathioneSepharose™ 4B (Amersham Pharmacia) according to the manufacturers'protocols.

(20) Preparation of GCUD1 Specific Antibody

Polyclonal antibody against GCUD1 was purified from the serum. Proteinsfrom cells transfected with plasmids expressing Flag-tagged GCUD1 wereseparated by 10% SDS-PAGE and immunoblotted with anti-GCUD1 or anti-Flagantibody. HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology,Santa Cruz, Calif.) or HRP-conjugated anti-Flag antibody served as thesecondary antibody, respectively, for ECL Detection System (AmershamPharmacia Biotech, Piscataway, N.J.). Immunoblotting with the anti-GCUD1antibody showed a 47 kD band of FLAG-tagged GCUD1, which pattern wasidentical to that detected with the anti-FLAG antibody.

(21) Statistical Analysis

The data were subjected to analysis of variance (ANOVA) and theScheffé's F test.

(22) Preparation of Peptides

9 mer and 10 mer peptides of RNF43, CXADRL1, or GCUD1 that bind toHLA-A24 or HLA-A*0201 molecule were predicted with the aid of bindingprediction software. These peptides were synthesized by Mimotopes, SanDiego, LA according to the standard solid phase synthesis method andpurified by reversed phase HPLC. The purity (>90%) and the identity ofthe peptides were determined by analytical HPLC and mass spectrometryanalysis, respectively. Peptides were dissolved in dimethylsulfoxide(DMSO) at 20 mg/ml and stored at −80° C.

(23) In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presentingcells (APCs) to induce CTL responses against peptides presented on HLA.DCs were generated in vitro as described elsewhere (Nukaya et al., Int JCancer 80: 92-7 (1999); Tsai et al., J Immunol 158: 1796-802 (1997)).Specifically, peripheral blood mononuclear cells (PBMCs) were isolatedfrom a healthy volunteer with HLA-A*0201 or HLA-A24 using Ficoll-Plaque(Pharmacia) solution, and monocyte fraction of PBMCs were separated byadherence to a plastic tissue culture flask (Becton Dickinson). Thismonocyte fraction was cultured for seven days in AIM-V medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS), 1000U/ml of GM-CSF (provided by Kirin Brewery Company), and 1000 U/ml ofIL-4 (Genzyme) to obtain DCs fraction. Candidate peptides were pulsedonto this DC enriched cell population at the concentration of 20 μg/ml,in the presence of 3 μg/ml of P2-microglobulin for 4 h at 20° C. inAIM-V. These peptide-pulsed antigen presenting cells were thenirradiated (5500 rad) and mixed at a 1:20 ratio with autologous CD8+ Tcells, obtained by positive selection with Dynabeads M-450 CD8 (Dynal)and Detachabead (Dynal). These cultures were set up in 48-well plates(Corning); each well contained 1.5×10⁴ peptide-pulsed antigen presentingcells, 3×10⁵ CD8+ T cells and 10 ng/ml of IL-7 (Genzyme) in 0.5 ml ofAIM-V with 2% AS. Three days later, these cultures were supplementedwith IL-2 (CHIRON) to a final concentration of 20 IU/ml. On day 7 and14, the T cells were further restimulated with the autologouspeptide-pulsed antigen presenting cells which were prepared each time inthe same manner as described above. Lymphoid cells in the culture on day21 were harvested and tested for cytotoxicity against peptide-pulsedTISI or T2 cells.

(24) CTL Expansion

Cultured lymphoid cells with proved significant cytotoxicity againstpeptide-pulsed TISI or T2 were further expanded in culture using amethod similar to that described by Riddell, et al. (Walter et al., NEngl J Med 333:1038-1044, 1995; Riddell et al., Nature Med. 2:216-23(1996)). 5×10⁴ of lymphoid cells were resuspended in 25 ml of AIM-Vsupplemented with 5% AS containing 25×10⁶ irradiated (3300 rads) PBMC,5×10⁶ irradiated (8000 rads) EHM cells, and 40 ng/ml of anti-CD3monoclonal antibody (Pharmingen). One day after initiating the cultures,120 IU/ml of IL-2 were added to the cultures. The cultures comprisedfresh AIM-V supplemented with 5% AS and 30 IU/ml of IL-2 on days 5, 8and 11.

(25) Establishment of CTL Clones

Some of the lymphoid cells with potent cytotoxicity were used to obtainCTL clones. The cell suspensions were diluted to densities of 0.3, 1,and 3 CTLs/lymphoid cells per well in 96 round-bottom microtiter plate(Nalge Nunc International). These cells were cultured in 150 μl/well ofAIM-V supplemented with 5% AS containing 7×10⁴ cells/well of allogenicPBMCs, 1×10⁴ cells/well of EHM, 30 ng/ml of anti-CD3 antibody, and 125U/ml of IL-2. 10 days later, 50 μl/well of IL-2 was added to the mediumto a final concentration of 125 U/ml. Cytotoxic activity of culturedCTLs was tested on day 14, and CTL clones were expanded using the samemethod as described above.

(26) Cytotoxicity Assay

Target cells were labeled with 100 μCi of Na₂ ⁵¹CrO₄ (Perkin Elmer LifeSciences) for 1 h at 37° C. in a CO₂ incubator. When peptide-pulsedtargets were used, target cells were incubated with the addition of 20μg/ml of the peptide for 16 h at 37° C. before the labeling with Na₂⁵¹CrO₄. Target cells were rinsed and mixed with effectors at a finalvolume of 0.2 ml in round-bottom microtiter plates. The plates werecentrifuged (4 minutes at 800×g) to increase cell-to-cell contact andplaced in a CO₂ incubator at 37° C. After 4 h of incubation, 0.1 ml ofthe supernatant was collected from each well and the radioactivity wasdetermined with a gamma counter. In case of evaluating cytotoxicityagainst target cells that endogenously express RNF43, CXADRL1, or GCUD1,the cytolytic activity was tested in the presence of a 30-fold excess ofunlabeled K562 cells to reduce any non-specific lysis due to NK-likeeffectors. Antigen specificity was confirmed by the cold targetinhibition assay, which utilized unlabeled TISI or T2 cells that werepulsed with peptide (20 μg/ml for 16 h at 37° C.) to compete for therecognition of ⁵¹Cr-labeled HT29 or SNU475 cells. The MHC restrictionwas examined by blocking assay, measuring the inhibition of thecytotoxicity with anti-HLA-class I (W6/32) antibody and anti-HLA-classII antibody, or anti-CD4 antibody and anti-CD8 antibody (DAKO).

The percentage of specific cytotoxicity was determined by calculatingthe percentage of specific ⁵¹Cr-release by the following formula: {(cpmof the test sample release−cpm of the spontaneous release)/(cpm of themaximum release−cpm of the spontaneous release)}×100. Spontaneousrelease was determined by incubating the target cells alone in theabsence of effectors, and the maximum release was obtained by incubatingthe targets with 1N HCl. All determinants were done in duplicate, andthe standard errors of the means were consistently below 10% of thevalue of the mean.

2. Results (1) Identification of Two Novel Human Genes, CXADRL1 andGCUD1, Commonly Up-Regulated in Gastric Cancers

By means of a genome-wide cDNA microarray containing 23040 genes,expression profiles of 20 gastric cancers were compared withcorresponding non-cancerous mucosae. Among commonly up-regulated genesdetected in the microarray analysis, a gene with an in-house accessionnumber of A5928 corresponding to an EST, Hs.6658 of UniGene cluster, wasfound to be over-expressed in a range between 4.09 and 48.60 (FIG. 1 a).Since an open reading frame of this gene encoded a protein approximately37% identical to that of CXADR (coxsackie and adenovirus receptor), thisgene was dubbed CXADRL1 (coxsackie and adenovirus receptor like 1).CXADRL1 was also up-regulated in 6 of 6 colorectal cancer cases and 12out of 20 HCC cases. Furthermore, a gene with an in-house accessionnumber of C8121, corresponding to KIAA0913 gene product (Hs.75137) ofUniGene cluster was also focused due to its significantly enhancedexpression in nine of twelve gastric cancer tissues compared with thecorresponding non-cancerous gastric mucosae by microarray (FIG. 1 b).This gene with the in-house accession number C8121 was dubbed GCUD1(up-regulated in gastric cancer). GCUD1 was also up-regulated in 5 of 6colorectal cancer cases, 1 out of 6 HCC cases, 1 out of 14 lung cancer(squeamous cell carcinoma) cases, 1 out of 13 testicular seminomascases. To clarify the results of the cDNA microarray, expression ofthese transcripts in gastric cancers was examined by semi-quantitativeRT-PCR to confirm an increased expression of CXADRL1 in all of the 10tumors (FIG. 1 c) and elevated expression of GCUD1 in seven of ninecancers (FIG. 1 d).

(2) Isolation and Structure of a Novel Gene CXADRL1

Multiple-tissue Northern-blot analysis using a PCR product of CXADRL1 asa probe revealed the expression of a 3.5-kb transcript in testis andovary (FIG. 2 a). Since A5928 was smaller than the gene detected on theNorthern-blot, 5′RACE experiments were carried out to determine theentire coding sequence of the CXADRL1 gene. The putative full-lengthcDNA consisted of 3423 nucleotides, with an open reading frame of 1296nucleotides (SEQ ID NO: 1) encoding a 431-amino-acid protein (SEQ ID NO:2) (GenBank Accession number: AB071618). The first ATG was flanked by asequence (CCCGGGATGA) (SEQ ID NO: 70) that was consistent with theconsensus sequence for the initiation of translation in eukaryotes, withan in-frame stop codon upstream. Using the BLAST program to search forhomologies in the NCBI (the National Center for BiotechnologyInformation) databases, a genomic sequence with the GenBank accessionnumber AC068984 was identified, which sequence had been assigned tochromosomal band 3q13. Comparison of the cDNA and the genomic sequencerevealed that CXADRL1 consisted of 7 exons (FIG. 2 b).

A search for protein motifs using the Simple Modular ArchitectureResearch Tool (SMART) revealed that the predicted protein contained twoimmunogloblin domains (codons 29-124 and 158-232) and a transmembranedomain (codons 246-268), suggesting that CXADRL1 might belong to theimmunogloblin super family.

(3) Effect of CXADRL1 on Cell Growth

A colony-formation assay was performed by transfecting NIH3T3 cells witha plasmid expressing CXADRL1 (pcDNA3.1myc/His-CXADRL1). Cellstransfected with pcDNA3.1myc/His-CXADRL1 produced markedly more coloniesthan mock-transfected cells (FIG. 3 a). To further investigate thisgrowth-promoting effect of CXADRL1, NIH3T3 cells that stably expressedexogenous CXADRL1 were established (FIG. 3 b). The growth rate ofNIH3T3-CXADRL1 cells was significantly higher than that of parentalNIH3T3 cells in culture media containing 10% FBS (FIG. 3 c).

(4) Suppression of CXADRL1 Expression in Human Gastric Cancer Cells byAntisense S-oligonucleotides

Six pairs of control and antisense S-oligonucleotides corresponding toCXADRL1 were transfected into MKN-1 gastric cancer cells, which hadshown the highest level of CXADRL1 expression among the examined sixgastric cancer cell lines. Six days after transfection, viability oftransfected cells was measured by MTT assay. Viable cells transfectedwith antisense S-oligonucleotides (CXADRL1-AS4 or -AS5) were markedlyfewer than those transfected with control S-oligonucleotides (CXADRL1-S4or -S5) (FIG. 4). Consistent results were obtained in three independentexperiments.

(5) Construction of Plasmids Expressing CXADRL1 siRNAs and Their Effecton the Growth of Gastric Cancer Cells

Plasmids expressing various CXADRL1-siRNA were constructed and examinedfor their effect on CXADRL1 expression. Among the constructed siRNAs,psiH1BX-CXADRL7 significantly suppressed the expression of CXADRL1 inSt-4 cells (FIG. 5A). To test whether the suppression of CXADRL1 mayresult in growth suppression of colon cancer cells, St-4 cells weretransfected with psiH1BX-CXADRL7 or mock vector. The number of viablecells transfected with psiH1BX-CXADRL7 was fewer than the number ofviable control cells (FIGS. 5B and 5C).

(6) Preparation of Anti-CXADRL1 Antibody

To examine the expression and explore the function of CXADRL1, antiserumagainst CXADRL1 was prepared. Immunoblotting with anti-CXADRL1 detecteda 50 kD band of FLAG-tagged CXADRL1, which was almost identical by sizeto that detected with anti-FLAG antibody (FIG. 6).

(7) Identification of CXADRL1-Interacting Protein by Yeast Two-HybridScreening System

To clarify the function of CXADRL1, CXADRL1-interacting proteins weresearched using yeast two-hybrid screening system. Among the positiveclones identified, C-terminal region of nuclear AIP1 (atriphininteracting protein 1) interacted with CXADRL1 in the yeast cells thatwere simultaneously transformed with pAS2.1-CXADRL1 and pACT2-AIP1 (FIG.7). The positive clones contained codons between 808 and 1008,indicating that the responsible region for the interaction with AIP1 iswithin this region.

(8) Prediction of Candidate Peptides for CTLs Derived from CXADRL1

Table 1 shows the candidate peptides (SEQ ID NOs: 115-154) in order ofhigh binding affinity. Forty peptides in total were selected andexamined as described below.

TABLE 1 Prediction of candidate peptides derived from CXADRL1 Ranksequence Score Position HLA-A*0201 9 mer 1 YLWEKLDNT 1314.7 176 2LLLLSLHGV 1006.2 11 3 INLNVIWMV 49.262 53 4 WMVTPLSNA 37.961 59 5CLVNNLPDI 23.995 120 6 SLHGVAASL 21.362 15 7 VIIIFCIAL 18.975 252 8LINLNVIWM 14.69 52 9 AVLPCTFTT 13.993 40 10 ALSSGLYQC 11.426 207 11VMSRSNGSV 11.101 384 12 SIFINNTQL 10.868 104 13 KVHRNTDSV 10.437 327 14RIGAVPVMV 9.563 413 15 NIGVTGLTV 9.563 132 16 SIYANGTHL 9.399 356 17LLCSSEEGI 8.691 163 18 LLSLHGVAA 8.446 13 19 IIFCIALIL 7.575 254 20TMPATNVSI 7.535 97 HLA-A*0201 10 mer 1 YLWEkLDNTL 3344 176 2 LINLnVIWMV280.45 52 3 ALSSgLYQCV 104.33 207 4 ALININVIWM 62.845 51 5 ILLCsSEEGI32.155 162 6 VLPCtFTTSA 32.093 41 7 LLLSIHGVAA 31.249 12 8 SIYAnGTHLV30.603 356 9 QLSDtGTYQC 20.369 111 10 GLYQcVASNA 15.898 211 11PLLLISLHGV 13.022 10 12 IQVArGQPAV 11.988 32 13 FINNtQLSDT 10.841 106 14LVPGqHKTLV 10.346 364 15 NLPDiGGRNI 8.555 124 16 VLVPpSAPHC 8.446 140 17AVIIiECIAL 7.103 251 18 VIIIfCIAL 5.609 252 19 ILGAfFYWRS 5.416 261 20GLTVIVPPSA 4.968 137

(9) Stimulation of T Cells and Establishment of CTL Clones UsingCandidate Peptides

Lymphoid cells were cultured using these candidate peptides derived fromCXADRL1 in the manner described in “Materials and Methods”. Resultinglymphoid cells showing detectable cytotoxic activity were expanded, andCTL clones were established. CTL clones were propagated from the CTLlines described above using the limiting dilution methods. A CTL cloneinduced with CXADRL1-207 (ALSSGLYQC) (SEQ ID NO: 124) showed highercytotoxic activities against the target pulsed with peptides than to thetargets not pulsed with any peptide. Cytotoxic activity of this CTLclone is shown in FIG. 8. This CTL clone had very potent cytotoxicactivity against the peptide-pulsed target without showing any cytotoxicactivity against the non-pulsed target.

(10) Cytotoxic Activity Against Tumor Cell Lines Endogenously ExpressingCXADRL1 as a Target

The CTL clones raised against predicted peptides were examined for theirability to recognize and kill tumor cells endogenously expressingCXADRL1. FIG. 9 shows the results of CTL Clone 75 raised againstCXADRL1-207 (ALSSGLYQC) (SEQ ID NO: 124). CTL Clone 75 showed potentcytotoxic activity against SNU475 that expresses CXADRL1 and HLA-A*0201,however, not against MKN74 that expresses CXADRL1 but not HLA-A*0201,and SNU-C4 that expresses HLA-A*0201 but not CXADRL1.

(11) Specificity of the Established CTLs

Cold target inhibition assay was performed to confirm the specificity ofCXADRL1-207 CTL Clone. SNU475 cells labeled with ⁵¹Cr were used as a hottarget, while T2 cells pulsed with CXADRL1-207 (SEQ ID NO: 124) wereused without ⁵¹Cr labeling as a cold target. Specific cell lysis againstSNU475 cells was significantly inhibited, when T2 pulsed withCXADRL1-207 (SEQ ID NO: 124) was added in the assay at various ratios(FIG. 10). The results are indicated as the percentage of specific lysisat an E/T ratio of 20.

In the interest of clone CXADRL1-207 CTL, to examine itscharacteristics, antibodies against HLA-Class I, HLA-Class II, CD4, andCD8 were tested for their capacity to inhibit its cytotoxic activity.The cytotoxicity of the CTL clone against SNU475 cells was significantlyinhibited when anti-HLA-Class I antibody and anti-CD8 antibody were used(FIG. 11), indicating that the CTL clone recognizes the CXADRL1 derivedpeptide in an HLA-Class I and CD8 dependent manner.

(12) Induction of CTLs by Anchor-Modified peptides (CXADRL1)

The newly defined epitope, CXADRL1-9 mer-207 (SEQ ID NO: 124) had arelatively low binding score. Thus, to increase the binding with MHCclass I molecule, altered peptides were developed from the CXADRL1-9mer-207.

HLA-A*0201 allele-binding peptides frequently are nonamers. Position twoand nine of the peptides are considered to be primary anchor residuesthat bind to the HLA class I peptide-binding cleft alone but do notcontact with TCR. According to the HLA-A2 antigen motif previouslyreported by Smith et al. (Smith et al., Mol Immunol 35: 1033-43 (1998)),leucine (Leu) and isoleucine (Iso) have proven to be an optimal anchorresidue at position 2 that enhance the binding affinity of the peptidefor the HLA-A*0201 molecule. Similarly, valine (Val) at position 9 isalso preferred for nonamer peptides. Based on these knowledge, twoanchor-modified altered peptides of CXADRL1-9 mer-207 with amino acidsubstitutions to contain the optimal HLA-A*0201 allele-binding aminoacids as anchor residues to increase the binding affinity to HLA-A*0201molecules were designed and synthesized.

TABLE 2 Anchor modified altered peptides; modification ofIGSF11CXADRL1-9mer-207 peptide with increased binding affinity forHLA-A*0201 molecule Amino Acid Binding Peptide Sequence Score* SEQ ID NOwild-type ALSSGLYQC 11.4 124 anchor-modified-1 ALSSGLYQV 49.0 195anchor-modified-2 ALSSGLYQL 159.8 196 *The binding score of thesealtered peptides were calculated by the BIMAS's epitope predictionalgorithm.

The binding score of these peptides were calculated using BIMAS'sepitope prediction algorithm as previously described, and both of thealtered peptides showed higher HLA-A*0201-binding scores compared to thewild-type CXADRL1 peptide. These anchor-modified peptides were testedfor their ability to elicit CTLs, and moreover, whether the induced CTLsrecognize not only the altered peptide but also the parental CXADRL1-9mer-207 peptide on T2. After CTL induction with CXADRL1-9V(SEQ IDNO:195) and CXADRL1-9L (SEQ ID NO:196), CTL line 5 and CTL clone 69 wereobtained by the stimulation with CXADRL1-9V peptide shown in FIGS. 12A12B. These CTLs cross-reacted with the wild-type CXADRL1-9 mer-207peptide on T2 cells, and also killed tumor cell line SNU475 thatendogenously express naturally processed CXADRL1 derived peptide, asmeasured by ⁵¹Cr release assay shown in FIG. 12C.

Several published studies have shown the utility of such alteredpeptides. Peptides having high affinity for an MHC class I molecule arepresented on the cell surfaces for a longer time than those with lowaffinity. Therefore, peptides with increased affinity for MHC class Imolecule have been considered to have increased potential to induceT-cell-mediated immune responses (Keogh et al., J Immunol 167: 787-96(2001); Tourdot et al., J Immunol 159: 2391-8 (1997); Sette et al., JImmunol 153: 5586-92 (1994)). The binding score calculated by theBIMAS's prediction software of CXADRL1-9 mer-207 (SEQ ID NO: 124) was11.4, relatively low compared to viral antigens or other TAAs (Bendnareket al., J Immunol 147: 4047-53 (1991); Sette et al, J Immunol 153:5586-92 (1994)). To increase immunogenicity of the native epitopepeptide, altered peptides were designed and synthesized that have theanchor residue at position 2 or 9 replaced with optimized amino acidsfor HLA-A*0201 (Vierboom et al., J Immunother 21: 399-408 (1998); Irvineet al., Cancer Res 59: 2536-40 (1999); Dyall et al., J Exp Med 188:1553-61 (1998); Muller et al., J Immunol 147: 1329-97 (1991)). CTLsraised against CXADRL1-9V (SEQ ID NO:195) with a binding score of 49responded not only to the CXADRL1-9V peptide but also to the parentalpeptide CXADRL1-9 mer-207 on T2 cells. Furthermore, these CTLs inducedby the GCUD1-9V peptide also recognized the native peptide naturallyprocessed by tumor cell. However, many recent studies indicate thatincreased affinity for MHC class I does not necessarily correlate withincreased peptide immunogenicity or an ability to stimulate CTL (Clay etal., J Immunol 162: 1749-55 (1999); Dionne et al., Cancer ImmunoImmunother 52: 199-206 (2003); Slansky et al., Immunity, 13: 529-38(2000); Sloan-Lancaster et al., Annu Rev Immunol 14: 1-27 (1996); Yanget al., J Immunol 169: 531-9 (2002); Trojan et al., Cancer Res 61:4761-5 (2001); Dionne et al, Cancer Immuno Immunother 53: 307-14(2004)). Alteration of peptide ligands can result in a generation ofpeptides with dramatically different phenotypes of the T cells andsometimes act as partial agonists or even as antagonists in course of Tcell activation or TCR signal transduction. It cannot be decided whetherCTLs stimulated with altered peptide work more efficiently as effectorsthan CTLs elicited by native peptide in vivo. However, in thisexperiment, an altered peptide, CXADRL1-9V, that activates a certainpart of TCR repertoire recognizing the naturally processed wild-typeepitope peptide presented by tumor cells was defined. A furtherimportant point is that this altered peptide could elicit native peptidespecific CTLs more frequently and abundantly than the wild-type peptide.Native peptide specific CTLs were generated in three of the fourindividuals upon stimulations with this altered peptide, whereas thewild-type peptide induced CTLs in only one healthy volunteer. Thisimproved induction of CTLs implicate some advantages in clinicalapplication, and provides an option in clinical investigations. In otherwords, the potentiality of anchor-modified peptide was demonstrated.

(13) Expression and Characterization of Novel Human Gene GCUD1

Multi-tissue Northern-blot analysis using GUCD1 cDNA as a probe showed a5.0-kb transcript that was specifically expressed in testis, ovary, andbrain (FIG. 13). Although the nucleotide sequence of KIAA0913 (GenBankAccession Number: XM-014766), corresponding to GCUD1, consisted of 4987nucleotides, RT-PCR experiments using testis, ovary, and cancer tissuesrevealed a transcript that consisted of 4987 nucleotides containing anopen reading frame of 1245 nucleotides (SEQ ID NO: 3) (GenBank AccessionNumber: AB071705). Furthermore, the genomic sequence corresponding toGUCD1 was searched in genomic databases to find a draft sequenceassigned to chromosomal band 7p14 (GenBank Accession Number:NT_(—)007819). Comparison between the cDNA sequence and the genomicsequence revealed that the GUCD1 gene consisted of 8 exons.

(14) Subcellular Localization of GCUD1

The entire coding region corresponding to GCUD1 was cloned intopCDNA3.1myc/His vector and the construct was transiently transfectedinto COS7 cell. Immunocytochemical staining of the COS7 cell revealedthat the tagged-GCUD1 protein was present in the cytoplasm (FIG. 14).

(15) Effect of GCUD1 on Cell Growth

To analyze the effect of GCUD1 on cell growth, a colony-formation assaywas conducted by transfecting NIH3T3 cells with a plasmid expressingGCUD1 (pcDNA3.1myc/His-GCUD1). Compared with a control plasmid(pcDNA3.1myc/His-LacZ), pcDNA3.1myc/His-GCUD1 induced markedly morecolonies in NIH3T3 cells (FIG. 15). This result was confirmed by threeindependent experiments.

(16) Growth Suppression of Gastric Cancer Cells by AntisenseS-oligonucleotides Designated to Reduce Expression of GCUD1

To test whether the suppression of GCUD1 may result in cell death ofgastric cancer cells, various antisense S-oligonucleotides designed tosuppress the expression of GCUD1 were synthesized. Six days aftertransfection of the respective antisense S-oligonucleotides, viabilityof transfected cells was measured by MTT assay. Viable cells transfectedwith antisense S-oligonucleotides (GCUD1-AS5 or -AS8) were markedlyfewer than those transfected with control S-oligonucleotides (GCUD1-S5or -S8) in MKN-28 cells (FIG. 16). This result was confirmed by threeindependent experiments. Similar result was observed with MKN-1 cells.

(17) Preparation of Anti-GCUD1 Antibody

To examine the expression and explore the function of GCUD1, antiserumagainst GCUD1 was prepared. Recombinant protein of GCUD1 was extractedand purified from bacterial cells expressing GST-GCUD1 fusion protein(FIG. 17). The recombinant protein was used for immunization of threerabbits. Immunoblotting with anti-GCDU1 sera but not pre-immune serashowed a 47 kD band of FLAG-tagged GCUD1, which was almost identical bysize to that detected with anti-FLAG antibody (FIG. 18).

(18) Prediction of Candidate peptides for CTLs Derived from GCUD1

Table 3 (GCUD1) shows the candidate peptides (SEQ ID NOs: 155-194) inorder of high binding affinity. Forty peptides in total were selectedand examined as described below.

TABLE 3 Prediction of candidate peptides derived from GCUD1 Ranksequence Score Position HLA-A*0201 9 mer 1 SIFKPFIFV 369.77 303 2WLWGAEMGA 189.68 75 3 IMISRPAWL 144.26 68 4 LLGMDLVRL 83.527 107 5FIFVDDVKL 49.993 308 6 VCIDSEFFL 31.006 265 7 KPFIFVDDV 25.18 306 8IVDRDEAWV 22.761 159 9 TLRDKASGV 21.672 257 10 KMDAEHPEL 21.6 196 11ALDVIVSLL 19.653 126 12 YAQSQGWWT 19.639 207 13 KLRSTMLEL 13.07 367 14YLIVDRDEA 11.198 157 15 AAPPSYCFV 7.97 3 16 GMDLVRLGL 6.171 109 17KVTEGVRCI 6.026 179 18 CIDSEFFLT 4.517 266 19 TVQTMMNTL 4.299 250 20EMGANEHGV 3.767 80 HLA-A*0201 10 mer 1 FIFVdDVKLV 374.37 308 2LIVDrDEAWV 366.61 158 3 FLTTaSGVSV 319.94 272 4 TMLEIEKQGL 234.05 371 5ALLGmDLVRL 181.79 106 6 AIMIsRPAWL 59.775 67 7 GVCIdSEFFL 59.628 264 8KLVPkTQSPC 17.388 315 9 FNFSeVFSPV 14.682 220 10 YISIdQVPRT 10.841 56 11GEGEfNFSEV 10.535 216 12 WAAEkVTEGV 8.927 175 13 VLPQnRSSPC 8.446 281 14AAAPpSYCFV 7.97 2 15 TMMNtLRDKA 6.505 253 16 EVGDIFYDCV 5.227 397 17AEMGaNEHGV 5.004 79 18 GLVVfGKNSA 4.968 20 19 QLSLtTKMDA 4.968 190 20RSIFkPFIFV 4.745 302

(19) Stimulation of T Cells and Establishment of CTL Clones UsingCandidate Peptides

Lymphoid cells were cultured using the above candidate peptides of (18)derived from GCUD1 in the manner described under the item of “Materialsand Methods”. Resulting lymphoid cells showing detectable cytotoxicactivity were expanded, and CTL clones were established. CTL clones werepropagated from the CTL lines described above using the limitingdilution methods. CTL clones induced with GCUD1-196 (KMDAEHPEL) (SEQ IDNO: 164) and GCUD1-272 (FLTTASGVSV) (SEQ ID NO: 177) showed highercytotoxic activities against the target pulsed with peptides than thetargets not pulsed with any peptide. Cytotoxic activity of these CTLclones is shown in FIG. 19. Each CTL clone had very potent cytotoxicactivity against the peptide-pulsed target without showing any cytotoxicactivity against the non-pulsed target.

(20) Cytotoxic Activity Against Tumor Cell Lines Endogenously ExpressingGCUD1 as a Target

The CTL clones raised against predicted peptides were examined for theirability to recognize and kill tumor cells endogenously expressing GCUD1.FIG. 20 shows the results of CTL Clone 23 raised against GCUD1-196 (SEQID NO: 164). CTL Clone 23 showed potent cytotoxic activity againstSNU475 which expresses GCUD1 and HLA-A*0201, however, not against MKN45that expresses GCUD1 but not HLA-A*0201.

(21) Specificity of Established CTLs

Cold target inhibition assay was also performed to confirm thespecificity of GCUD1-196 CTL Clone. SNU475 cells labeled with ⁵¹Cr wereused as a hot target, while T2 cells pulsed with GCUD1-196 were usedwithout ⁵¹Cr labeling as a cold target. Specific cell lysis againstSNU475 cells was significantly inhibited, when T2 pulsed with GCUD1-196(SEQ ID NO: 164) was added in the assay at various ratios (FIG. 21). Theresults are indicated as the percentage of specific lysis at an E/Tratio of 20.

In the interest of the GCUD1-196 (SEQ ID NO: 164) CTL clone, to examineits characteristics, antibodies against HLA-Class I, HLA-Class II, CD4,and CD8 were tested for their capacity to inhibit its cytotoxicactivity. The cytotoxicity of the CTL clone against SNU475 cells wassignificantly inhibited by the use of anti-HLA-Class I antibody andanti-CD8 antibody (FIG. 22), indicating that the CTL clone recognizesthe GCUD1 derived peptide in an HLA-Class I and CD8 dependent manner.

(22) Induction of CTLs by Anchor-Modified Peptides (GCUD1)

The newly defined epitope, GCUD1-196 (SEQ ID NO: 164) showed relativelylow binding score. Therefore, to increase its binding ability with MHCclass I molecule, altered peptides were made from the GCUD1-196 peptide.

As mentioned above, HLA-A*0201 allele-binding peptides are frequentlynonamers whose amino acids at position two and nine are consideredprimary anchor residues that solely bind to the HLA class Ipeptide-binding cleft but do not contact with TCR. According to theHLA-A2 antigen motif previously reported by Smith et al. (Smith et al.,Mol Immunol 35: 1033-43 (1998)), leucine (Leu) and isoleucine (Iso) haveproven to be an optimal anchor residue at position 2 that enhance thebinding affinity of the peptide for the HLA-A*0201 molecule. Similarly,valine (Val) at position 9 was also preferred for nonamer peptides.

Thus, two anchor-modified altered peptides of GCUD1-196 with amino acidsubstitutions that contain the optimal HLA-A*0201 allele-binding aminoacids as anchor residues to increase the binding affinity to HLA-A*0201molecules were designed and synthesized.

TABLE 4 Anchor modified altered peptides; modification of GCUD1-196wild-type peptide with increased binding affinity for HLA-A*0201molecule Amino Acid Binding Peptide Sequence Score** SEQ ID NO wild-typeKMDAEHPEL 21.6 164 anchor-modified-1 KLDAEHPEL 29.9 197anchor-modified-2 KMDAEHPEV 70.3 198 anchor-modified-3 KLDAEHPEV 97.3199 (N.S.*) *N.S.: Not synthesized due to its hydrophobicity of itsamino acid sequence. *Binding Score: High scores indicate high affinityto MHC class I molecules. The binding scores of these altered peptideswere calculated by the BIMAS's epitope prediction algorithm.

The binding score of these peptides were calculated based on the BIMAS'sepitope prediction algorithm as previously described, and all threealtered peptides received higher HLA-A*0201-binding scores compared tothe wild-type GCUD1 peptide. These anchor-modified peptides were testedfor their ability to elicit CTLs, and moreover, whether the induced CTLsrecognize the parental GCUD1-196 peptide on T2 in addition to thealtered peptides. After CTL induction with GCUD1-9V (SEQ ID NO:198) andGCUD1-2L (SEQ ID NO: 197), CTL line 3 and CTL clone 16 were obtained viathe stimulation with GCUD1-9V peptide shown in FIGS. 23A and 23B. TheseCTLs cross-reacted with the wild-type GCUD1-196 peptide on T2 cells, andalso killed tumor cell line SNU475 that endogenously express naturallyprocessed GCUD1-196 peptide, as measured by ⁵¹Cr release assay shown inFIG. 23C.

Notably, GCUD1-196 specific CTLs were generated in three of the fourHLA-A*0201 positive individuals by GCUD1-9V peptide (SEQ ID NO: 198),whereas GCUD1-196 wild-type peptide specific responses were observed inone of the four individuals when GCUD1-196 was used for the CTLinduction.

The binding score of GCUD1-196 (SEQ ID NO: 164) wild-type peptidecalculated by the BIMAS's prediction software was 21.6, relatively lowcompared to those of viral antigens or other TAAs (Bendnarek et al., JImmunol 147: 4047-53 (1991); Sette et al., J Immunol 153: 5586-92(1994)). To increase immunogenicity of this native epitope peptide,altered peptides with anchor residue modification at position 2 or 9,replacement to optimized amino acids for HLA-A*0201, were designed andsynthesized (Vierboom et al., J Immunother 21: 399-408 (1998); Irvine etal., Cancer Res 59: 2536-40 (1999); Dyall et al., J Exp Med 188: 1553-61(1998); Muller et al, J Immunol 147: 1329-97 (1991)). CTLs raisedagainst GCUD1-9V peptide (SEQ ID NO: 198) with a binding score of 70.3responded not only to the GCUD1-9V peptide but also to the parentalwild-type peptide GCUD1-196 on T2 cells. Furthermore, these CTLs inducedby the GCUD1-9V peptide also recognized the native peptide naturallyprocessed by tumor cell. However, as mentioned above, many recentstudies indicated that increased affinity for MHC class I did notnecessarily correlate with increased peptide immunogenicity or abilityto stimulate CTL (Clay et al., J Immunol 162: 1749-55 (1999); Dionne etal., Cancer Immuno Immunother 52: 199-206 (2003); Slansky et al.,Immunity 13: 529-38 (2000); Sloan-Lancaster and Allen, Annu Rev Immunol14: 1-27 (1996); Yang et al., J Immunol 169: 531-9 (2002); Trojan etal., Cancer Res, 61: 4761-5 (2001); Dionne et al., Cancer ImmunoImmunother 53: 307-14 (2004)); and alteration of peptide ligands canresult in a generation of peptides with dramatically differentphenotypes of the T cells, and sometimes act as partial agonists or evenas antagonists in course of T cell activation or TCR signaltransduction. Therefore, it cannot be decided whether CTLs stimulatedwith altered peptide work more efficiently as effectors than CTLselicited by native peptide in vivo. However, in this experiment, analtered peptide, GCUD1-9V, that activates a certain part of TCRrepertoire recognizing the naturally processed wild-type epitope peptidepresented by tumor cells was defined. More important is that thisaltered peptide elicited native peptide specific CTLs more frequentlyand abundantly than the wild-type peptide. Three of the four individualsgenerated the native peptide specific CTLs by stimulations with thisaltered peptide, whereas the wild-type peptide induced CTLs in only onehealthy volunteer. This evidence may implicate some advantages inclinical application, and an option in clinical investigations wasprovided by the present invention. In other words, a potentialanchor-modified altered peptide was provided.

The amino acid sequences of GCUD1-196 (SEQ ID NO:164) and GCUD1-9V (SEQID NO:198) were subjected to BLAST's homology analysis. No peptide withhigh homology derived from any other known molecule was detected. Theseresults support the fact that the identified peptides are GCUD1-specificand have little chance to possess cross-reactivity against other knownmolecules. This also suggests the possibility of the use of thesepeptides in clinical applications without unwanted adverse effects.

(23) Identification of Gene FLJ20315 Commonly Up-Regulated in HumanColon Cancer

Expression profiles of 11 colon cancer tissues were compared withnon-cancerous mucosal tissues of the colon corresponding thereto usingthe cDNA microarray containing 23040 genes. According to this analysis,expression levels of a number of genes that were frequently elevated incancer tissues were compared to corresponding non-cancerous tissues.Among them, a gene with an in-house accession number of B4469corresponding to an EST (FLJ20315), Hs. 18457 in UniGene cluster, wasup-regulated in the cancer tissues compared to the correspondingnon-cancerous mucosae at a magnification range between 1.44 and 11.22(FIG. 24 a). FLJ20315 was also up-regulated in 6 out of 18 gastriccancer cases, 12 out of 20 HCC cases, 11 out of 22 lung cancer(adenocarcinoma) cases, 2 out of 2 testicular seminomas cases and 3 outof 9 prostate cancer cases. To clarify the results of the microarray,the expression of these transcripts in additional colon cancer sampleswere examined by semi-quantitative RT-PCR to confirm the increase ofFLJ20315 expression in 15 of the 18 tumors (FIG. 24 b).

(24) Expression and Structure of RNF43

Additional homology searches of the sequence of FLJ20315 in publicdatabases using BLAST program in National Center for BiotechnologyInformation identified ESTs including XM_(—)097063, BF817142, and agenomic sequence with a GenBank Accession Number NT_(—)010651 assignedto chromosomal band 17pter-p13.1. As a result, an assembled sequence of5345 nucleotides containing an open reading frame of 2352 nucleotides(SEQ ID NO: 5) encoding a 783-amino-acid protein (SEQ ID NO: 6) (GenBankAccession Number: AB081837) was obtained. The gene was dubbed RNF43(Ring finger protein 43). The first ATG was flanked by a sequence(AGCATGC) that agreed with the consensus sequence for initiation oftranslation in eukaryotes, and by an in-frame stop codon upstream.Comparison of the cDNA and the genomic sequence revealed that this geneconsisted of 11 exons. Northern-blot analysis using human adultMultiple-Tissue Northern-blots with a PCR product of RNF43 as a probefailed to detect any band (data not shown). However, a 5.2 kb-transcriptwas detected to be expressed in fetal lung and fetal kidney using ahuman fetal tissue Northern-blot with the same PCR product as a probe(FIG. 25 a). A search for protein motifs with the Simple ModularArchitecture Research Tool (SMART) revealed that the predicted proteincontained a Ring finger motif (codons 272-312) (FIG. 25 b).

(25) Subcellular Localization of myc-Tagged RNF43 Protein

To investigate the subcellular localization of RNF43 protein, a plasmidexpressing myc-tagged RNF43 protein (pDNAmyc/His-RNF43) was transientlytransfected into COS7 cells. Western-blot analysis using extracts fromthe cells and anti-myc antibody revealed a 85.5-KDa band correspondingto the tagged protein (FIG. 26 a). Subsequent immunohistochemicalstaining of the cells with the same antibody indicated the protein to bemainly present in the nucleus (FIG. 26 b). Similar subcellularlocalization of RNF43 protein was observed in SW480 human colon cancercells.

(26) Effect of RNF43 on Cell Growth

A colony-formation assay was conducted by transfecting NIH3T3 cells witha plasmid expressing RNF43 (pcDNA-RNF43). Cells transfected withpcDNA-RNF43 produced significantly more number of colonies than controlcells (FIG. 27 a). Increased activity of colony formation by RNF43 wasalso shown in SW480 cells wherein the endogenous expression of RNF43 wasvery low (data not shown). To further investigate this growth-promotingeffect, COS7 cells that stably express exogenous RNF43 (COS7-RNF43) wereestablished (FIG. 27 b). The growth rate of COS7-RNF43 cells wassignificantly higher than that of COS7-mock cells in culture mediacontaining 10% FBS (FIG. 27 c).

(27) Growth Suppression of Colon Cancer Cells by AntisenseS-oligonucleotides Designated to Reduce Expression of RNF43

To test whether the suppression of RNF43 expression may result in growthretardation and/or cell death of colon cancer cells, five pairs ofcontrol and antisense S-oligonucleotides corresponding to RNF43 weresynthesized and transfected into LoVo colon cancer cells, which show ahigher level of RNF43 expression among the examined 11 colon cancer celllines. Among the five antisense S-oligonucleotides, RNF43-AS1significantly suppressed the expression of RNF43 compared to controlS-oligonucleotides (RNF43-S1) 12 hours after transfection (FIG. 28 a).Six days after transfection, number of surviving cells transfected withRNF43-AS1 was significantly fewer than those transfected with RNF43-S1suggesting that the suppression of RNF43 expression reduced growthand/or survival of transfected cells (FIG. 28 b). Consistent resultswere obtained in three independent experiments.

(28) Construction of Plasmids Expressing RNF43 siRNAs and Their Effecton Growth of Colon Cancer Cells

In mammalian cells, small interfering RNA (siRNA) composed of 20 or21-mer double-stranded RNA (dsRNA) with 19 complementary nucleotides and3′ terminal complementary dimmers of thymidine or uridine, has beenrecently shown to have a gene specific gene silencing effect withoutinducing global changes in gene expression. Therefore, plasmidsexpressing various RNF43-siRNAs were constructed to examine their effecton RNF43 expression. Among the various RNF43-siRNAs, psiH1BX-RNF16-4 andpsiH1BX-RNF1834 significantly suppressed the expression of RNF43 inSNUC4 cells (FIG. 29A). To test whether the suppression of RNF43 resultsin growth suppression of colon cancer cells, SNUC4 cells weretransfected with psiH1BX-RNF16-4, psiH1BX-RNF1834 or mock vector. Inline with the data of antisense S-oligonucleotides, the number of viablecells transfected with psiH1BX-RNF16-4 or psiH1BX-RNF1834 was fewer thanthe number of viable control cells (FIGS. 29B and 29C).

(29) Secretion of Flag-tagged RNF43 Protein in Culture Media of COS7Cells with Exogenous Flag-tagged RNF43 Protein

Since a search for protein motifs with amino acid sequence of RNF43using Simple Modular Architecture Research Tool (SMART) predicted asignal peptide and a ring finger domain, secretion of the RNF43 proteinwas examined. Plasmid expressing Flag-tagged RNF43 (pFLAG-RNF43) orMyc-tagged RNF43 (pcDNA3.1-Myc/His-RNF43), or mock vector wastransfected into COS7 cells, and the cells were cultured in mediasupplemented with 0.5% of bovine calf serum for 48 h. Western-blotanalysis with anti-Flag antibody or anti-Myc antibody detected secretedFlag-tagged RNF43 or Myc-tagged protein in the media containing cellstransfected with pFLAG-RNF43 or pcDNA3.1-Myc/His-RNF43, respectively,but not in the media containing cells with mock vector (FIGS. 30A and30B).

(30) Effect of Cultured Media of Cells Transfected with pFLAG-RNF43 onNIH3T3 Cells

Since exogenous expression of RNF43 conferred growth promoting effect onNIH3T3 cells, secreted Flag-tagged RNF43 was examined whether it alsoexerts a proliferative effect on NIH3T3 cells. NIH3T3 cells werecultured without the change of media, or with conditioned media ofmock-transfected cells, or cells transfected with pFlag-RNF43. Asexpected, NIH3T3 cells showed a significantly higher growth rate whencultured in in conditioned media of cells transfected with eitherpFlag-RNF43 or pcDNA3.1-Myc/His-RNF43 compared to those cultured inconditioned media of non-treated cells or mock-vector transfected cells(FIGS. 31A and 31B). These data suggest that RNF43 may exert its growthpromoting effect in an autocrine manner.

(31) Preparation of Recombinant amino- and carboxyl-Terminal RNF43Protein

To generate a specific antibody against RNF43, a plasmid expressingNus-tagged RNF43 protein was constructed (FIG. 32A). Upon transformationof the plasmid into E. coli BL21trxB(DE3)pLysS cell, production of arecombinant protein in the bacterial extract with the expected size wasobserved by SDS-PAGE (FIGS. 32B and 32C).

(32) Identification of RNF43-Interacting Proteins by Yeast Two-HybridScreening System

To clarify the oncogenic mechanism of RNF43, RNF43-interacting proteinswere searched using yeast two-hybrid screening system. Among theidentified positive clones, NOTCH2 or STRIN interacted with RNF43 bysimultaneous transformation of an yeast cell with pAS2.1-RNF43 andpACT2-NOTCH2 (FIG. 33B), or pAS2.1-RNF43 and pACT2-STRIN (FIG. 34B). Theregions responsible for the interaction in NOTCH2 and STRIN areindicated in FIGS. 33A and 34A, respectively.

(33) Prediction of HLA-A24 Binding peptides Derived from RNF43

The amino acid sequence of RNF43 was scanned for peptides with a lengthof 9 or 10 amino acids which peptides bind to HLA-A24 using the bindingprediction software. Table 5 shows the predicted peptides (SEQ ID NOs:71-90) in order of high binding affinity. Twenty peptides in total wereselected and examined as described below.

TABLE 5 Predicted RNF43 peptides binding to HLA-A24(http://bimas.dcrtnih.gov/cgi-bin/ molbio/ken_parker_comboform) Start AAsequence Binding Start AA sequence Binding position (9 mers) affinity*¹position (10 mers) affinity RNF43-331 SYQEPGRRL 360 RNF43-449 SYCTERSGYL200 RNF43-350 HYHLPAAYL 200 RNF43-350 HYHLPAAYLL 200 RNF43-639 LFNLQKSSL30 RNF43-718 CYSNSQPVWL 200 RNF43-24 GFGRTGLVL 20 RNF43-209 IFVIILASVL36 RNF43-247 RYQASCRQA 15 RNF43-313 VFNITEGDSF 15 RNF43-397 RAPGEQQRL 14RNF43-496 TFCSSLSSDF 12 RNF43-114 RAPRPCLSL 12 RNF43-81 KLMQSHPLYL 12RNF43-368 RPPRPGPFL 12 RNF43-54 KMDPTGKLNL 9 RNF43-45 KAVIRVIPL 12RNF43-683 HYTPSVAYPW 8 RNF43-721 NSQPVWLCL 10 RNF43-282 GQELRVISCL 4In the table, start position indicates the location of amino acids fromthe N-terminus of RNF43.

(34) Stimulation of T Cells Using the Predicted Peptides

CTLs against these peptides derived from RNF43 were generated accordingto the method described in the above “Materials and Methods”. ResultingCTLs showing detectable cytotoxic activity were expanded, and CTL lineswere established.

The cytotoxic activities of CTL lines induced by 9 mer-peptide (SEQ IDNOs: 71-80) stimulation are shown in Table 6.

TABLE 6 Cytotoxicity of CTL lines (9 mer) Cytotoxicity Start AA Binding× 20 × 2 Established position sequences affinity Pep(+) Pep(−) Pep(+)Pep(−) CTL clones RNF43-331 SYQEPGRRL 360.0 2% 1% 0% 0% RNF43-350HYHLPAAYL 200.0 26% 17% 5% 4% RNF43-639 LFNLQKSSL 30.0 42% 33% 7% 5% 1RNF43-24 GFGRTGLVL 20.0 8% 9% 1% 2% RNF43-247 RYQASCRQA 15.0 71% 82% 28%16% RNF43-397 RAPGEQQRL 14.4 41% 32% 15% 15% RNF43-114 RAPRPCLSL 12.023% 26% 6% 9% RNF43-368 RPPRPGPFL 12.0 1% 0% 0% 0% RNF43-45 KAVIRVIPL12.0 NE RNF43-721 NSQPVWLCL 10.0 68% 0% 26% 0% 13 NE:No establishment ofCTL linesCTL lines stimulated with RNF43-350 (HYHLPAAYL) (SEQ ID NO: 72),RNF43-639 (LFNLQKSSL) (SEQ ID NO: 73), and RNF43-721 (NSQPVWLCL) (SEQ IDNO: 80) showed higher cytotoxic activities on the target that werepulsed with peptides than on the target that was not pulsed with any ofthe peptides. Starting from these CTLs, one CTL clone was establishedwith RNF43-639 and 13 CTL clones were established with RNF43-721.

The CTL line stimulated with RNF43-721 showed a potent cytotoxicactivity on the peptide-pulsed target without showing any significantcytotoxic activity on the target that was not pulsed with any of thepeptides (FIG. 35).

The results obtained by examining the cytotoxic activity of CTL linesstimulated with the 10 mer-peptides (SEQ ID NOs: 81-90) are shown inTable 7.

TABLE 7 Cytotoxicity of CTL lines (10 mer) Cytotoxicity Start AA Binding× 20 × 2 Established position sequences affinity Pep(+) Pep(−) Pep(+)Pep(−) CTL clones RNF43-449 SYCTERSGYL 200.0 1% 1% 0% 0% RNF43-350HYHLPAAYLL 200.0 NE RNF43-718 CYSNSQPVWL 200.0 NE RNF43-209 IFVLILASVL36.0 Not synthesis RNF43-313 VFNLTEGDSF 15.0 Not synthesis RNF43-496TFCSSLSSDF 12.0 8% 9% 0% 0% RNF43-81 KLMQSHPLYL 12.0 10% 5% 2% −3%RNF43-54 KMDPTGKLNL 9.6 5% 2% 0% −1% RNF43-683 HYTPSVAYPW 8.4 0% 0% 0%−1% RNF43-282 GQELRVISCL 8.4 NE NE:No establishment of CTL linesCTL lines stimulated with RNF43-81 (KLMQSHPLYL) (SEQ ID NO: 87) orRNF43-54 (KMDPTGKLNL) (SEQ ID NO: 88) showed modest cytotoxic activityon the peptide-pulsed target compared with that on the target that wasnot pulsed with any of the peptides.

(35) Establishment of CTL Clones

CTL clones were propagated from the CTL lines described above using thelimiting dilution method. 13 CTL clones against RNF43-721 and 1 CTLclone against RNF43-639 were established (see Table 6 supra). Thecytotoxic activity of RNF43-721 CTL clones is shown in FIG. 36. Each CTLclone had a very potent cytotoxic activity on the peptide-pulsed targetwithout showing any cytotoxic activity on the target that was not pulsedwith any of the peptides.

(36) Cytotoxic Activity Against Colorectal Cancer Cell LinesEndogenously Expressing RNF43 as Target

The CTL clones raised against predicted peptides were examined for theirability to recognize and kill tumor cells that endogenously expressRNF43. FIG. 37 shows the results of CTL Clone 45 raised againstRNF43-721. CTL Clone 45 showed a potent cytotoxic activity on HT29 andWiDR both expressing RNF43 and HLA-A24. On the other hand, CTL Clone 45did not show any cytotoxic activity on either HCT116 (expressing RNF43but not HLA-A24) or TISI (expressing HLA-A24 but not RNF43). Moreover,CTL Clone 45 did not show any cytotoxic activity on irrelevant peptidepulsed TISI and SNU-C4 that express RNF43 but little HLA-A24 (data notshown).

(37) Characterization of Established CTLs

A cold target inhibition assay was performed to confirm the specificityof RNF43-721 CTL Clone. HT29 cells labeled with ⁵¹Cr were used as a hottarget, while TISI cells pulsed with RNF43-721 without ⁵¹Cr labelingwere used as a cold target. When TISI pulsed with RNF43-721 was added inthe assay at various ratios, specific cell lysis against the HT29 celltarget was significantly inhibited, (FIG. 38). This result is indicatedas the percentage of specific lysis at an E/T ratio of 20. To examinethe characteristics of the CTL clone raised against RNF43 peptide,antibodies against HLA-Class I, HLA-Class II, CD3, CD4, and CD8 weretested for their ability to inhibit the cytotoxic activity of the CTLclone. The cytotoxicity of the CTL clone on the WiDR cell target wassignificantly inhibited when anti-HLA-Class I, CD3, and CD8 antibodieswere used (FIG. 39). The result indicates that the CTL clone recognizesthe RNF43 derived peptide via HLA-Class I, CD3, and CD8.

(38) Homology Analysis of RNF43-721 Peptide

The CTL clones established against RNF43-721 showed a very potentcytotoxic activity. This result may indicate that the sequence ofRNF43-721 is homologous to the peptides derived from other moleculeswhich are known to sensitize human immune system. To exclude thispossibility, homology analysis of RNF43-721 was performed using BLAST.No sequence completely matching or highly homologous to RNF43-721 wasfound among the molecules listed in BLAST (Table 8).

TABLE 8 Homology analysis of RNF43-721 Identification(9/9) 0Identification(8/9) 0 Identification(7/9) 0 Identification(6/9) 2(BLAST)These results indicate that the sequence of RNF43-721 is unique andthere is little possibility for the CTL clones established withRNF43-721 to raise immunologic response to other molecules.

(39) Modification of RNF43-721 to Improve the Efficacy of EpitopePresentation

To improve the efficacy of peptide presentation, RNF43-721 peptide weremodified at amino acid alternations on the anchor site. The modificationwas expected to improve the binding affinity of the peptide to the HLAClass I molecule. Table 9 demonstrates a better binding affinity toHLA-A24 molecule of RNF43-721 with alternations of amino acids atposition 2 (SEQ ID NOs: 91 and 92).

TABLE 9 Predicted binding capacities of the peptides modified from theRNF43-721 native peptide Sequence Score Rank* NSQPVWLCL 10.08 10NFQPVWLCL 50.40 3 NYQPVWLCL 504.00 1 *In HLA-A24 restricted 9 merpeptides

(40) Prediction of HLA-A02 Binding Peptides Derived from RNF43

Table 10 shows candidate peptides (SEQ ID NOs: 87, and 93-111) in orderof high binding affinity.

TABLE 10 RNF43:Prediction of epitope peptides (HLA-A*0201) Starting Noposition Sequences Score 9 mer 1 60 KLNLTLEGV 274.3 2 8 QLAALWPWL 199.73 82 LMQSHPLYL 144.2 4 358 LLGPSRSAV 118.2 5 11 ALWPWLLMA 94.8 6 15WLLMATLQA 84.5 7 200 WILMTVVGT 40.1 8 171 KLMEFVYKN 34.5 9 62 NLTLEGVFA27.3 10 156 GLTWPVVLI 23.9 10 mer 11 81 KLMQSHPLYL 1521.5 12 357YLLGPSRSAV 1183.7 13 202 LMTVVGTIFV 469.6 14 290 CLHEFHRNCV 285.1 15 500SLSSDFDPLV 264.2 16 8 QLAAIWPWLL 160.2 17 11 ALWPWLLMAT 142.2 18 7LQLAALWPWL 127.3 19 726 WLCLTPRQPL 98.2 20 302 WLHQHRTCPL 98.2Twenty peptides in total were selected and examined as described below.

(41) Stimulation of T Cells Using Candidate Peptides

Lymphoid cells were cultured using the candidate peptides derived fromRNF43 according to the method described in the above “Materials andMethods”. Resulting lymphoid cells showing detectable cytotoxic activitywere expanded, and CTL lines were established. The cytotoxic activitiesof CTL lines induced by the stimulation using 9 mer-peptides (SEQ IDNOs: 93-102) are shown in Table 11.

TABLE 11 Cytotoxicity of CTL lines (HLA-A*0201 9 mer) Cytotoxicity(%)Binding × 20 × 2 Start position AA sequences affinity Pep(+) Pep(−)Pep(+) Pep(−) RNF43-60 KLNLTLEGV 274.3 −2.1 0.2 −1.6 0.0 RNF43-8QLAALWPWL 199.7 3.5 0.0 0.0 1.0 RNF43-82 LMQSHPLYL 144.2 1.7 1.2 0.0−0.4 RNF43-358 LLGPSRSAV 118.2 −0.4 −0.7 0.0 −0.8 RNF43-11 ALWPWLLMA94.8 90.2 1.5 45.4 1.3 RNF43-15 WLLMATLQA 84.5 −0.2 0.0 −0.4 −0.9RNF43-200 WILMTVVGT 40.1 Not Synthesis RNF43-171 KLMEFVVKN 34.5 2.6 0.01.1 −0.5 RNF43-62 NLTLEGVFA 27.3 Not Synthesis RNF43-156 GLTWPVVLI 23.9−0.4 0.7 −0.5 −0.3 NE:No establishment of CTL linesCTL lines induced with RNF43-11-9 (ALWPWLLMA) (SEQ ID NO: 97) showedhigher cytotoxic activities on the target pulsed with peptides than onthe target that was not pulsed with any of the peptides. Starting fromthese CTLs, four CTL clone were established with RNF43-11-9. The CTLline stimulated with RNF43-11-9 showed a potent cytotoxic activity onthe peptide-pulsed target without showing any significant cytotoxicactivity on the target that was not pulsed with any of the peptides(FIG. 40A).

The results of examination on the cytotoxic activity of CTL linesinduced with the 10 mer-peptides (SEQ ID NOs: 87, and 103-111) are shownin Table 12.

TABLE 12 Cytotoxicity of CTL lines (HLA-A*0201 10 mer) Cytotoxicity(%)Binding × 20 × 2 Start position AA sequences affinity Pep(+) Pep(−)Pep(+) Pep(−) RNF43-81 KLMQSHPLYL 1521.5 18.0 27.6 6.3 8.3 RNF43-357YLLGPSRSAV 1183.7 18.2 15.4 3.7 3.0 RNF43-202 LMTVVGTTFV 469.6 NotSynthesis RNF43-290 CLHEFHRNCV 285.1 9.6 9.7 2.7 3.7 RNF43-500SLSSDFDPLV 264.2 NE RNF43-8 QLAATWPWLL 160.2 6.7 9.0 1.1 1.3 RNF43-11ALWPWLLMAT 142.2 91.5 27.1 40.5 4.3 RNF43-7 LQLAALWPWL 127.3 NERNF43-726 WLCLTPRQPL 98.2 NE RNF43-302 WLHQHRTCPL 98.2 7.4 6.1 1.5 2.2NE:No establishment of CTL linesCTL lines induced with RNF43-11-10 (ALWPWLLMAT) (SEQ ID NO: 108) showeda higher cytotoxic activity on the peptide-pulsed target than on thetarget that was not pulsed with any of the peptides (FIG. 40B).

(42) Establishment of CTL Clones

CTL clones were propagated from the CTL lines described above using thelimiting dilution method. Four CTL clones against RNF43-11-9 wereestablished (see Table 11 supra). The cytotoxic activity of RNF43peptides-derived CTL clones is shown in FIGS. 41A and 41B. Each CTLclone had a very potent cytotoxic activity on the peptide-pulsed targetwithout showing any cytotoxic activity on the target that was not pulsedwith any of the peptides.

(43) Cytotoxic Activity Against Colorectal Cancer Cell LinesEndogenously Expressing RNF43 as Targets

The CTL clones raised against the predicted peptides were examined fortheir ability to recognize and kill tumor cells that endogenouslyexpress RNF43. FIGS. 42A and 42B show the results obtained for the CTLclones raised against RNF43 derived peptides. The CTL Clones showed apotent cytotoxic activity on DLD-1 which expresses RNF43 and HLA-A*0201,but none on HT29 which expresses RNF43 but not HLA-A*0201.

(44) Specificity of CTL Clones

A cold target inhibition assay was performed to confirm the specificityof RNF43-5-11(9 mer) CTL Clone. HCT116 cells labeled with ⁵¹Cr were usedas a hot target, while T2 cells pulsed with RNF43-5 without ⁵¹Crlabeling were used as a cold target. Specific cell lysis of the HCT-116cell target was significantly inhibited, when T2 pulsed with RNF43-5 wasadded in the assay at various ratios (FIG. 43).

INDUSTRIAL APPLICABILITY

The expression of novel human genes CXADRL1 and GCUD1 is markedlyelevated in gastric cancer as compared to non-cancerous stomach tissues.On the other hand, the expression of novel human gene RNF43 is markedlyelevated in colorectal cancers as compared to non-cancerous mucosaltissues. Accordingly, these genes may serve as a diagnostic marker ofcancer and the proteins encoded thereby may be used in diagnostic assaysof cancer.

The present inventors have also shown that the expression of novelprotein CXADRL1, GCUD1, or RNF43 promotes cell growth whereas cellgrowth is suppressed by antisense oligonucleotides or small interferingRNAs corresponding to the CXADRL1, GCUD1, or RNF43 gene. These findingssuggest that each of CXADRL1, GCUD1, and RNF43 proteins stimulateoncogenic activity. Thus, each of these novel oncoproteins is a usefultarget for the development of anti-cancer pharmaceuticals. For example,agents that block the expression of CXADRL1, GCUD1, or RNF43, or preventits activity may find therapeutic utility as anti-cancer agents,particularly anti-cancer agents for the treatment of gastric andcolorectal cancers. Examples of such agents include antisenseoligonucleotides, small interfering RNAs, and antibodies that recognizeCXADRL1, GCUD1, or RNF43.

Furthermore, the present inventors have shown that CXADRL1 interactswith AIP1. It is expected that the cell proliferating activity ofCXADRL1 is regulated by its binding to AIP1. Thus, agents that inhibitthe activity of the formation of a complex composed of CXADRL1 and AIP1may also find utility in the treatment and prevention of cancer,specifically colorectal, lung, gastric, and liver cancers.Alternatively, the present inventors have shown that RNF43 interactswith NOTCH2 or STRIN. It is expected that the cell proliferatingactivity of RNF43 is regulated by its binding to NOTCH2 or STRIN. Thus,agents that inhibit the activity of the formation of a complex composedof RNF43 and NOTCH2 or STRIN may also find utility in the treatment andprevention of cancer, specifically colorectal, lung, gastric, and livercancers.

The present inventions have also shown that the peptides, derived fromthe amino acid sequence of CXADRL1, GCUD1 or RNF43 protein, stimulate Tcells and induce cytotoxic T cells. The peptides are useful as vaccineto induce anti-tumor immunity.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

1. A substantially pure polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide that comprises the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, inserted, and/or added and that has a biological activity equivalent to a protein consisting of the amino acid sequence of SEQ ID NO: 2; and (c) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of any one of SEQ ID NO:
 2. 2. A substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO: 124, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 124 in which at least one anchor residue is modified.
 3. The polypeptide of claim 2, wherein the polypeptide comprises amino acid sequence of 124 or
 195. 4. The polypeptide of claim 3, wherein the polypeptide consists of amino acid sequence of 124 or
 195. 5. A pharmaceutical composition for treating or preventing a cancer, said composition comprising a pharmaceutically effective amount of polypeptide selected from the group of (a)-(c), or a polynucleotide encoding the polypeptide: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or fragments thereof, (b) a polypeptide that comprises the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, inserted, and/or added and that has a biological activity equivalent to a protein consisting of the amino acid sequence of SEQ ID NO: 2 or fragments thereof; and (c) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or fragments thereof. as an active ingredient, and a pharmaceutically acceptable carrier.
 6. The pharmaceutical composition of claim 5, which comprises a pharmaceutically effective amount of polypeptide comprising the amino acid sequence of SEQ ID NO: 124, or polypeptide comprising the amino acid sequence of SEQ ID NO: 124 in which at least one anchor residue is modified.
 7. The pharmaceutical composition of claim 6, wherein the polypeptide comprises amino acid sequence of 124 or
 195. 8. The pharmaceutical composition of claim 7, wherein the polypeptide consists of amino acid sequence of 124 or
 195. 